Selective degradation of wild-type dna and enrichment of mutant alleles using nuclease

ABSTRACT

The present disclosure provides methods for preparing a target mutant nucleic acid for subsequent enrichment relative to a wild-type nucleic acid using nucleases that have a substantially higher activity on double stranded DNA versus single stranded DNA or RNA. The present disclosure also includes methods for enriching a target mutant nucleic acid and for preparing unmethylated/methylated nucleic acids of interest for subsequent enrichment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.15/739,301, filed Dec. 22, 2017, issued on Aug. 15, 2023 as U.S. Pat.No. 11,725,230, which is a national phase application under 35 USC § 371of PCT Application No. PCT/US2016/039167, filed Jun. 24, 2016, whichclaims benefit of U.S. provisional application Ser. No. 62/183,854,filed Jun. 24, 2015, the contents of each of which are herebyincorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number R21CA175542 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

A commonly encountered situation in genetic analysis entails the need toidentify a low percent of variant DNA sequences (‘minority alleles’) inthe presence of a large excess of non-variant sequences (‘majorityalleles’). Examples for such situations include the following:identification and sequencing of a few mutated alleles in the presenceof a large excess of normal (wild-type) alleles, a commonly encounteredsituation iisancer (for example, identification of tumor-circulating DNAin blood or in urine of cancer patients (or abnormal DNA in peoplesuspected of having cancer) in the presence of a large excess ofwild-type alleles); identification of a few methylated alleles in thepresence of a large excess of unmethylated alleles (or vice-versa) inepigenetic analysis; identification and genotyping of a few fetal DNAsequences circulating in the maternal blood where a large excess ofmaternal DNA sequences are also present; identification of emergingmutated strains in infectious agents (bacteria or viruses); and variantsequence detection for crop development.

While reliable high throughput screening methods for germline orhigh-prevalence somatic mutations have been described, detection oflow-prevalence somatic mutations in tumors with heterogeneity, stromalcontamination, or in bodily fluids is still problematic. And yet, theclinical significance of identifying these mutations is very importantin several situations. For example, in lung adenocarcinoma, low-levelEGFR mutations that cannot be identified by regular sequencing canconfer positive response to tyrosine kinase inhibitors or drugresistance. Mutations in plasma, useful as biomarkers for earlydetection of cancer or cancer response to treatment, cannot be sequencedusing conventional methods due to the high excess of wild-type allelesoriginating from normal tissues. Additionally, mutations in tumors withfrequent stromal contamination, such as pancreatic or prostate cancer,can be ‘masked’ by presence of wild-type alleles, thus requiringlaborious micro-dissection or resulting in missing mutations altogether.

Beyond cancer, low levels of target DNA in the presence of high levelsof non-target DNA occurs in many other fields and applications. Forexample, the detection of a small number of fetal alleles withinmaternal alleles is especially important for prenatal diagnosis duringearly stages in pregnancy where fetal alleles comprise a low fraction.An especially interesting application to this end is the fact that fetalalleles are substantially hypomethylated compared to maternal alleles.Thus, there is a general need to develop techniques that allow foridentification of low-level minority alleles (for example, mutated orhypo/hypermethylated alleles) in the presence of high level non-variantmajority alleles.

SUMMARY

The present disclosure relates to a novel development (Nuclease-assistedMutation Enrichment, NaME) that results to preferential cleavage ofwild-type nucleic acids, thereby allowing for subsequent enrichment ofmutated target sequences of interest. The mutation-enriched sequencescan then be screened using any currently available methods foridentifying mutations, such as Sanger Sequencing, high resolutionmelting (HRM), etc.

Accordingly, some aspects of the disclosure provide a method forpreparing a target mutant nucleic acid for subsequent enrichmentrelative to a wild-type nucleic acid. The method comprises subjecting anucleic acid sample comprising a double-stranded wild-type nucleic acidand a double-stranded target nucleic acid suspected of containing amutation to a condition that destabilizes the double stranded wild-typeand target mutant nucleic acids; contacting the destabilized doublestranded wild-type and target mutant nucleic acids with a pair ofoligonucleotide probes, one of which is complementary to the wild-typenucleic acid top strand and the other is complementary to the wild-typenucleic acid bottom strand, to permit hybridization of the probes totheir corresponding sequences on the wild-type and target mutant nucleicacids thereby forming complementary wild-type-probe duplexes on top andbottom strands, and partially complementary target mutant-probeduplexes, wherein at least one of the probes overlaps a sequence on thetarget nucleic acid containing the suspected mutation; and exposing thecomplementary wild-type-probe duplexes and the partially complementarytarget mutant-probe duplexes to a double strand-specific nuclease (DSN),wherein the DSN cleaves the complementary wild-type-probe duplexes butnot the partially complementary target mutant-probe duplexes.

Some aspects of the disclosure provide a method for preparing a targetmutant nucleic acid for subsequent enrichment relative to a wild-typenucleic acid comprising exposing a nucleic acid sample comprising adouble-stranded wild-type nucleic acid and a double-stranded targetnucleic acid suspected of containing a mutation to a doublestrand-specific nuclease (DSN) and a pair of oligonucleotide probes, oneof which is complementary to the wild-type nucleic acid top strand andthe other is complementary to the wild-type nucleic acid bottom strand,to create a reaction mixture, wherein at least one of the probesoverlaps a sequence on the target nucleic acid containing the suspectedmutation; and subjecting the reaction mixture to a condition thatdestabilizes the double stranded wild-type and target mutant nucleicacids to permit hybridization of the probes to their correspondingsequences on the wild-type and target mutant nucleic acids therebyforming complementary wild-type-probe duplexes on top and bottomstrands, and partially complementary target mutant-probe duplexes,wherein the DSN cleaves the complementary wild-type-probe duplexes butnot the partially complementary target mutant-probe duplexes.

In some embodiments, the condition that destabilizes the double strandedwild-type and mutant nucleic acids to permit hybridization of the probesto their corresponding sequences on the wild-type and mutant nucleicacids comprises addition of an organic solvent and/or an increase intemperature combined with a thermostable DSN.

Some aspects of the disclosure provide a method for preparing a targetmutant nucleic acid for subsequent enrichment relative to a wild-typenucleic acid comprising exposing a nucleic acid sample comprising adouble-stranded wild-type nucleic acid and a double-stranded targetnucleic acid suspected of containing a mutation to a pair ofoligonucleotide probes, one of which is complementary to the wild-typenucleic acid top strand and the other is complementary to the wild-typenucleic acid bottom strand, to create a reaction mixture, wherein atleast one of the probes overlaps a sequence on the target nucleic acidcontaining the suspected mutation; subjecting the reaction mixture to adenaturing temperature to permit denaturation of the wild-type nucleicacid and the target mutant nucleic acid; reducing the temperature of thereaction mixture to permit formation of complementary wild-type-probeduplexes on top and bottom strands and partially complementary targetmutant-probe duplexes; and exposing the reaction mixture to a doublestrand-specific nuclease (DSN), wherein the DSN cleaves thecomplementary wild-type-probe duplexes but not the partiallycomplementary target mutant-probe duplexes.

In some embodiments, the method is used to prepare an unmethylatedtarget nucleic acid of interest for subsequent enrichment, and whereinprior to implementing the NaME protocol described herein on the reactionmixture, the nucleic acid sample is treated with sodium bisulfite andone of the oligonucleotide probes is complementary to top strand of themethylated nucleic acid of interest, while the other oligonucleotideprobe is complementary to the bottom strand of the methylated nucleicacid of interest.

In some embodiments, the method is used to prepare a methylated targetnucleic acid of interest for subsequent enrichment, and wherein prior toimplementing the NaME protocol described herein on the reaction mixture,the nucleic acid sample is treated with sodium bisulfite and one of theoligonucleotide probes is complementary to top strand of theunmethylated nucleic acid of interest, while the other oligonucleotideprobe is complementary to the bottom strand of the unmethylated nucleicacid of interest.

In some embodiments, the method is used to prepare both an unmethylatedtarget nucleic acid of interest and a methylated target nucleic acid ofinterest for subsequent enrichment, wherein the method comprises: (i) apair of oligonucleotide probes, one of which is complementary to topstrand of the methylated form of the unmethylated target nucleic acid ofinterest, while the other is complementary to the bottom strand of themethylated form of the unmethylated target nucleic acid of interest,(ii) a pair of oligonucleotide probes, one of which is complementary totop strand of the unmethylated form of the methylated target nucleicacid of interest, while the other is complementary to the bottom strandof the unmethylated form of the methylated target nucleic acid ofinterest; and wherein prior to implementing the NaME protocol describedherein on the reaction mixture, the nucleic acid sample is treated withsodium bisulfite.

In some embodiments, the method is used to prepare multiple targetnucleic acids of interest, some of which are methylated target nucleicacids of interest, and some of which are unmethylated target nucleicacids of interest, and the method comprises (i) a pair ofoligonucleotide probes, one of which is complementary to top strand ofthe methylated form of each unmethylated target nucleic acid ofinterest, while the other is complementary to the bottom strand of themethylated form of each unmethylated target nucleic acid of interest,(ii) a pair of oligonucleotide probes, one of which is complementary totop strand of the unmethylated form of each methylated target nucleicacid of interest, while the other is complementary to the bottom strandof the unmethylated form of each methylated target nucleic acid ofinterest.

Some aspects of the disclosure provide a method for preparing a targetmutant nucleic acid for subsequent enrichment relative to a wild-typenucleic acid, the method comprising the steps of: (a) exposing a nucleicacid sample comprising a double-stranded wild-type nucleic acid and adouble-stranded target nucleic acid suspected of containing a mutationto a thermostable double strand-specific nuclease (DSN) and a pair ofoligonucleotide probes, one of which is complementary to the wild-typenucleic acid top strand and the other is complementary to the wild-typenucleic acid bottom strand, to create a reaction mixture, wherein atleast one of the probes overlaps a sequence on the target nucleic acidcontaining the suspected mutation; (b) subjecting the reaction mixtureto a denaturing temperature to permit denaturation of the wild-typenucleic acid and the target mutant nucleic acid; and (c) reducing thetemperature to permit hybridization of the probes to their correspondingsequences on the wild-type and target mutant nucleic acids therebyforming complementary wild-type-probe duplexes on top and bottomstrands, and partially complementary target mutant-probe duplexes,wherein the DSN cleaves the complementary wild-type-probe duplexes butnot the partially complementary target mutant-probe duplexes.

In some embodiments, steps (b) and (c) are repeated for two or morecycles. In some embodiments, the reaction mixture further comprises anorganic solvent. In some embodiments, the denaturing temperature isbetween 65-85° C.

Furthermore, in some embodiments step (c) of reducing the temperature isperformed by applying a decreasing temperature gradient betweentemperatures that permit hybridization of probes having different Tm totheir corresponding sequences on the target DNA. For example, adecreasing temperature gradient from 67° C. to 64° C. can be appliedduring DSN digestion, to allow diverse probes that have distinct Tms of64-67° C. to hybridize effectively to their respective targets.(‘Touch-down NaME’). The temperature gradient can preferably rangebetween 2-20° C.

Some aspects of the disclosure provide a method for enriching a targetmutant nucleic acid, the method comprising the steps of: (a) preparingan amplification reaction mixture comprising: a double-strandedwild-type nucleic acid, a double-stranded target nucleic acid suspectedof containing a mutation, a thermostable double strand-specific nuclease(DSN), a pair of oligonucleotide probes, one of which is complementaryto the wild-type nucleic acid top strand and the other is complementaryto the wild-type nucleic acid bottom strand, wherein at least one of theprobes overlaps a sequence on the target nucleic acid containing thesuspected mutation and PCR amplification components; (b) subjecting thereaction mixture to a denaturing temperature to permit denaturation ofthe wild-type nucleic acid and the target mutant nucleic acid; (c)reducing the temperature to permit hybridization of the probes to theircorresponding sequences on the wild-type and target mutant nucleic acidsthereby forming complementary wild-type-probe duplexes on top and bottomstrands, and partially complementary target mutant-probe duplexes,wherein the DSN cleaves the complementary wild-type-probe duplexes butnot the partially complementary target mutant-probe duplexes; and (d)subjecting the reaction mixture to an amplification condition therebyenriching the uncleaved target mutant nucleic acid relative to thecleaved wild-type nucleic acid.

In some embodiments the amplification condition is such thatamplification is applied to the probes rather than the hybridizednucleic acid. In some embodiments, a purification step is appliedfollowing probe binding to top-and-bottom DNA target strands, eitherbefore or after DSN cleavage, to remove excess unbound probes. Thenfollowing DSN cleavage the uncut probes are amplified (instead ofamplifying the target DNA) and identified/quantified. Since probes thatbind WT DNA will have been selectively digested by DSN, the presence ofany given probe after amplification indicates a mutation under theregion covered by this probe.

In some embodiments, steps (b) and (c) are repeated for two or morecycles before executing step (d). In some embodiments, steps (b), (c)and (d) are repeated for two or more cycles. In some embodiments, thereaction mixture further comprises an organic solvent. In someembodiments, the denaturing temperature is between 65-85° C. In someembodiments, the primers used for PCR amplification have a meltingtemperature that is below the temperature applied in step (c). In someembodiments, the amplification condition is COLD-PCR.

Some aspects of the disclosure provide a method for preparingunmethylated nucleic acids of interest for subsequent enrichmentrelative to corresponding methylated nucleic acids comprising the stepsof: (a) ligating bisulfite-resistant adaptors to double stranded nucleicacids of interest; (b) subjecting the adaptor-linked nucleic acids tosodium bisulfite treatment and a nucleic acid amplification reaction toform double-stranded bisulfite-treated nucleic acids; (c) subjecting thebisulfite-treated nucleic acids to a temperature that permitspreferential denaturation of unmethylated nucleic acids while methylatednucleic acids remain double-stranded; (d) exposing the unmethylated andmethylated nucleic acids to double strand-specific nuclease (DSN) andconditions for optimal DSN activity, wherein the DSN cleaves themethylated double-stranded nucleic acids but not the unmethylatedsingle-stranded nucleic acids.

Some aspects of the disclosure provide a method for preparingunmethylated nucleic acids of interest for subsequent enrichmentrelative to corresponding methylated nucleic acids comprising the stepsof: (a) ligating bisulfite-resistant adaptors to double stranded nucleicacids of interest; (b) subjecting the adaptor-linked nucleic acids tosodium bisulfite treatment and a nucleic acid amplification reaction toform double-stranded bisulfite-treated nucleic acids; (c) subjecting thebisulfite-treated nucleic acids to a denaturing temperature that permitsdenaturation of both unmethylated and methylated nucleic acids to formunmethylated and methylated single stranded nucleic acids; (d) reducingthe temperature to permit preferential formation of methylated duplexes,but not unmethylated duplexes; and (e) exposing the unmethylated andmethylated nucleic acids to double strand-specific nuclease (DSN) andconditions for optimal DSN activity, wherein the DSN preferentiallycleaves the methylated duplexes but not the unmethylated single-strandednucleic acids.

Some aspects of the disclosure provide a method for preparing methylatednucleic acids of interest for subsequent enrichment relative tocorresponding unmethylated nucleic acids comprising the steps of: (a)ligating bisulfite-resistant adaptors to double stranded nucleic acidsof interest; (b) subjecting the adaptor-linked nucleic acids to sodiumbisulfite treatment and a nucleic acid amplification reaction to formdouble-stranded bisulfite-treated nucleic acids; (c) subjecting thebisulfite-treated nucleic acids to a temperature that permitspreferential denaturation of unmethylated nucleic acids while methylatednucleic acids remain double-stranded; and (d) exposing the unmethylatedand methylated nucleic acids to an exonuclease and conditions foroptimal exonuclease activity, wherein the exonuclease cleaves theunmethylated single-stranded nucleic acids but not the methylateddouble-stranded nucleic acids.

Some aspects of the disclosure provide a method for preparing methylatednucleic acids of interest for subsequent enrichment relative tocorresponding unmethylated nucleic acids comprising the steps of: (a)ligating bisulfite-resistant adaptors to double stranded nucleic acidsof interest; (b) subjecting the adaptor-linked nucleic acids to sodiumbisulfite treatment and a nucleic acid amplification reaction to formdouble-stranded bisulfite-treated nucleic acids; (c) subjecting thebisulfite-treated nucleic acids to a denaturing temperature that permitsdenaturation of both unmethylated and methylated nucleic acids to formunmethylated and methylated single stranded nucleic acids; (d) reducingthe temperature to permit preferential formation of methylated duplexes,but not unmethylated duplexes; and (e) exposing the unmethylated andmethylated nucleic acids to an exonuclease and conditions for optimalexonuclease activity, wherein the exonuclease preferentially cleaves theunmethylated single-stranded nucleic acids, but not the methylatedduplexes.

In some embodiments, the nucleic acid amplification reaction of step (b)is selected from the group consisting of: PCR; full COLD-PCR, fastCOLD-PCR; ice-COLD-PCR, temperature-tolerant COLD-PCR and limiteddenaturation time COLD-PCR. In some embodiments, the cleavedunmethylated single stranded nucleic acids and the uncleaved methylatedduplexes are subjected to an amplification condition using the bisulfiteresistant adaptors ligated in step (a). In some embodiments, theamplification condition is selected from the group consisting of: PCR,full COLD-PCR, fast COLD-PCR; ice-COLD-PCR, temperature-tolerantCOLD-PCR and limited denaturation time COLD-PCR. In some embodiments,naturally AT-rich sequences are removed prior to the sodium bisulfitetreatment.

In any of the foregoing methods, the probes are in a molar excess of100-fold to 1 billion-fold compared to the wild-type and target nucleicacids. In any of the foregoing methods, one of the probes overlaps asequence on the top strand of the target nucleic acid containing themutation, while the other probe overlaps a sequence on the bottom strandof the target nucleic acid containing the mutation and the two probespartially overlap each other. In any of the foregoing methods, eachprobe comprises a locked nucleic acid (LNA), peptide nucleic acid (PNA),xeno nucleic acid (XNA), nucleic acid with any known modified base orRNA. In any of the foregoing methods, the method is used to prepare twoor more different target mutant nucleic acids for subsequent enrichmentrelative to corresponding wild-type nucleic acids, and the methodfurther comprises one or more additional pairs of probes directed to thedifferent wild-type nucleic acids, wherein for each pair of probes, oneof the probes is complementary to the wild-type nucleic acid top strandand the other is complementary to the wild-type nucleic acid bottomstrand. In any of the foregoing methods, the nucleic acid samplecomprises genomic DNA or circulating DNA in urine, plasma or otherbodily fluids.

In any of the foregoing methods, the methods further comprise enrichingthe nucleic acids for regions of interest prior to implementing NaMEprotocol described herein as follows: contacting the nucleic acid samplewith bait oligonucleotides that bind to different nucleic acids ofinterest on top and bottom strands, permitting binding of the baitoligonucleotides to the nucleic acids of interest on top and bottomstrands, and isolating the bait oligonucleotides with the nucleic acidsof interest bound thereto from remaining nucleic acids. In someembodiments, the bait oligonucleotides are biotinylated at one end. Insome embodiments, the bait oligonucleotides are attached to beads.

In any of the foregoing methods, prior to implementing the NaME protocoldescribed herein, the nucleic acid sample is subjected to anamplification condition.

In any of the foregoing methods, the methods further comprise enrichingthe target mutant nucleic acid relative to the wild-type nucleic acid bysubjecting the reaction mixture with cleaved wild-type-probe duplexesand uncleaved target mutant nucleic acids to an amplification conditionthereby enriching the uncleaved target mutant nucleic acid relative tothe cleaved wild-type nucleic acid. In some embodiments, theamplification condition is selected from the group consisting of: PCR,COLD-PCR, ligation mediated PCR or COLD-PCR using common ligatedadaptors, multiplex PCR, and isothermal amplification.

In any of the foregoing methods, each probe can be optionally modifiedat the 3′ end to prevent polymerase extension.

In any of the foregoing methods, the methods further comprise enrichingthe target mutant nucleic acid relative to the wild-type nucleic acid bysubjecting the reaction mixture with cleaved wild-type-probe duplexesand uncleaved target mutant nucleic acids to a further DNA degradationcondition which hydrolyzes enzymatically the DSN-cleaved wild-type-probeduplexes, with the degradation initiated at the position of thecleavage.

In any of the foregoing methods, the methods further comprise analyzingthe reaction mixture with cleaved wild-type-probe duplexes and uncleavedtarget mutant nucleic acids using one or more of the methods selectedfrom the group consisting of: MALDI-TOF, HR-Melting,Di-deoxy-sequencing, Single-molecule sequencing, massively parallelsequencing (MPS), pyrosequencing, SSCP, RFLP, dHPLC, CCM, digital PCRand quantitative-PCR.

In some embodiments of any one of the provided methods, the DSN enzymeis a DNA guided or RNA guided enzyme. In some embodiments of any one ofthe provided methods, the enzyme is an RNA guided enzyme, e.g., Cas9. Insome embodiments of any one of the provided methods, the enzyme is a DNAguided enzyme, e.g., an Argonaute enzyme.

Some aspects of the disclosure provide a method for preparing a targetmutant nucleic acid for subsequent enrichment relative to a wild-typenucleic acid comprising subjecting a nucleic acid sample comprising adouble-stranded wild-type nucleic acid and a double-stranded targetnucleic acid suspected of containing a mutation to a condition thatdestabilizes the double stranded wild-type and target mutant nucleicacids; contacting the destabilized double stranded wild-type and targetmutant nucleic acids with a pair of oligonucleotide probes, one of whichis complementary to the wild-type nucleic acid top strand and the otheris complementary to the wild-type nucleic acid bottom strand, to permithybridization of the probes to their corresponding sequences on thewild-type and target mutant nucleic acids thereby forming complementarywild-type-probe duplexes on top and bottom strands, and partiallycomplementary target mutant-probe duplexes,

wherein at least one of the probes overlaps a sequence on the targetnucleic acid containing the suspected mutation, and wherein one or bothprobes comprise a locked nucleic acid (LNA), peptide nucleic acid (PNA),xeno nucleic acid (XNA), or a nucleic acid with any known modified baseor RNA which is capable of blocking PCR amplification; and subjectingthe complementary wild-type-probe duplexes on top and bottom strands,and partially complementary target mutant-probe duplexes to anamplification condition. The probe(s) that overlap the mutation positionact to block PCR amplification, e.g., acting as a clamp, for thewild-type top and bottom DNA strands, thereby inhibiting amplificationof the wild-type nucleic acid. When the probe duplexes with a partiallycomplementary target mutant sequence, it is less able to inhibit PCRamplification, thereby permitting selective amplification of the mutantnucleic acid as compared to the wild-type, without a need for a cleavingenzyme (e.g., DSN).

Each of the embodiments and aspects of the invention can be practicedindependently or combined. Also, the phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting. The use of “including”, “comprising”, or “having”,“containing”, “involving”, and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

These and other aspects of the inventions, as well as various advantagesand utilities will be apparent with reference to the DetailedDescription. Each aspect of the invention can encompass variousembodiments as will be understood.

All documents identified in this application are incorporated in theirentirety herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic the selective degradation of wild-typedouble-stranded DNA using duplex specific nuclease (DSN) and sequenceselective oligonucleotides (‘probes’) at elevated temperatures. A DNAdenaturation step prior to DSN action may optionally be applied in orderto generate single stranded DNA prior to probe binding and selectivewild-type degradation of both DNA strands. FIG. 1B shows the validationof NaME-based mutation enrichment as described in FIG. 1A via digitalPCR (ddPCR) for a mutated KRAS amplicon with 5% original mutationabundance (mutation abundance=fractional ratio of mutant to wild-typeDNA, expressed as a percentage). In this example, the DNA did notundergo a 95° C. denaturation step; the DNA, two probes, and DSN weremixed, and the temperature was elevated (67° C.) to de-stabilize theduplex and enable probe binding. A range of temperatures was applied toidentify the optimal temperatures for WT-specific digestion. FollowingDSN action, the DSN was inactivated via heating at 95° C. and dropletdigital PCR (ddPCR) was applied to the digested sample. ddPCR quantifiesthe mutation enrichment achieved by measuring fractional mutationabundance before and after DSN action.

FIG. 2 is a schematic showing the use of partially overlapping probes toprovide selectivity simultaneously on both DNA strands when binding amutation. Probes are preferably 3′-blocked to prevent subsequentpolymerase extension.

FIG. 3 is a schematic showing the use of duplex specific nuclease andpartially overlapping sequence selective oligonucleotides to enable theselective degradation of double-stranded, or denatured, DNA. With themutant sequence, there is limited complementarity between the top andbottom strand probes, which prohibits probe-to-probe interactions athigher temperatures.

FIG. 4 is a schematic showing wild-type allele degradation directly fromfragmented genomic DNA (i.e., not pre-amplified DNA). Note that amultiplex approach can be taken; thousands of probes can be usedsimultaneously on selected DNA targets of interest.

FIG. 5 shows the results of a single-plex assay showing the majorincrease in mutation abundance of mutational sequences following a DSNreaction directly on fragmented genomic DNA for a selected TP53 exon 8target sequence. The mutation abundance is quantified before and aftertreatment of the sample with DSN, using ddPCR. The mutation abundanceincreases only if both top and bottom probes are included in thereaction, in addition to DSN nuclease.

FIG. 6 shows the results of a duplex assay, showing the mutationabundance of a sample containing mutated KRAS and p53 at 5% or 0.3%original abundance, following a DSN reaction directly on fragmentedgenomic DNA.

FIG. 7A shows the results of a DSN reaction on genomic DNA for asingle-plex assay using DNA mutated at three different positions on KRASexon 2 in three different cell lines: H2009, A549, and HCT-15.

FIG. 7B is a table summarizing the mutation abundance found in a DSNreaction performed directly on fragmented genomic DNA via an 11-plexassay. The 11 mutated targets were formed using DNA from Horizon Dx. Inthe figure, column 1 shows the name of the target gene, columns 2 and 3represent the mutation position and mutation type, respectively, column4 shows the mutation abundance as derived via digital PCR when DSN isomitted; column 5 shows the mutation abundance when DSN is applied(NaME), column 6 represents the mutation abundance when both DSN and theprobes are omitted, and column 7 shows the expected mutation abundanceaccording to the Horizon manufacturer, without any treatment.

FIG. 8 is a schematic illustrating two approaches to selectivedegradation of wild-type DNA prior to targeted re-sequencing: one aftermultiplexed PCR (top), and one prior to PCR (bottom) using selectivebinding of DNA targets to beads. In the latter situation, the probesutilized are designed to partially overlap each other and bind both topand bottom DNA target strands. In addition, they are biotinylated. Theseprobes are first used without DSN to bind their targets and toimmobilize selected DNA targets to beads. The non-immobilized DNA isthen removed from the solution. The temperature then is then adjustedaccordingly, and NaME is applied as described above.

FIG. 9 is a schematic illustrating the ‘nuclease chain reaction’.Wild-type degradation is combined with denaturation cycles during theDSN digestion process resulting in improved discrimination betweenmutant and WT and, therefore, better mutation enrichment. Note that,following brief denaturation at 85° C. and cooling to 65° C., thereaction cycles again at 85° C. before substantial re-hybridization ofthe two strands can occur.

FIG. 10 is a schematic illustrating the PCR-NaME chain reaction whereboth PCR amplification and nuclease chain reaction operatesimultaneously on the sample, in a single tube. The successive cycles ofPCR synthesis and wild-type-specific degradation led to improvedenrichment of mutated sequences.

FIG. 11 is a schematic illustrating COLD-PCR-NaME. The successive cyclesof mutant-specific synthesis and wild-type-specific degradation enrichfor mutated sequences.

FIG. 12A is schematic illustrating mutation scanning using two or morelonger (non-overlapping) probes on opposite strands. Probes arepreferentially 3′-blocked to prevent polymerase extension, and maycontain modified bases, such as LNA, PNA, XNA, deoxy-inosinetriphosphate (dITP), or contain dUTP, or comprise RNA. In some cases,part of the probes can comprise one or more random nucleotides, so thatthe probe can be directed against a plurality of DNA targets. The totalcombined sequence under the two probes is interrogated during NaME: ifthere is a mutation anywhere under the two probes, it will preventstrand cutting.

FIG. 12B shows that the combination of denaturation and DSN for mutationscanning as described in 12A results in the preferential cutting ofwild-type DNA. A mutation present anywhere under the two probes resultsin the amplification of the mutated DNA during subsequent PCR or digitalPCR. The effects of probe length and concentration on mutationenrichment are depicted in the graphs.

FIG. 12C shows mutation scanning using two adjacent probes and DSN inNaME as described in 12A. The graph presents the average mutationenrichment-fold of each listed mutation.

FIG. 12D shows mutation scanning using two adjacent probes and DSN inNaME as described in 12A. The graph presents the average mutationenrichment-fold of each listed mutation.

FIG. 13 is a schematic representing the application of NaME with RNA orsingle-stranded DNA. Multiplexed wild-type nucleic acid degradation isused to enrich mutants prior to cDNA synthesis or PCR.

FIG. 14 is a schematic showing hypomethylation enrichment fromfragmented genomic DNA using probes designed to match the methylatedalleles on the target sequences.

FIG. 15 shows the enrichment of unmethylated DNA sequences andmethylation-sensitive temperature-tolerant-COLD-PCR followingpreferential DSN digestion of the sequences having a Tm higher than theTm of choice. This scheme is a genome-wide application and does notrequire the use of gene-specific probes or selection of targetsequences. The scheme enriches and amplifies hypo-methylated alleles ona genome-wide basis.

FIG. 16 depicts the process of using exonuclease-based temperaturefractionation of DNA fragments to remove lower Tm fragments prior tobisulfite conversion.

FIG. 17 is a flowchart showing the temperature-based elution of genomicDNA fragments by utilizing binding of DNA to magnetic beads.

DETAILED DESCRIPTION

In most applications involving detection of low-prevalence somaticmutations, the mutant alleles are detected following a polymerase chainreaction (PCR) step that amplifies both mutant and wild-type alleles.Methods have also been described to preferentially amplify the mutatedalleles over wild-type alleles (e.g., co-amplification at lowerdenaturation temperature or COLD-PCR and improved and completeenrichment COLD PCR or ice-COLD-PCR; Li J, Wang L, Mamon H, Kulke M H,Berbeco R, Makrigiorgos G M. Replacing PCR with COLD-PCR enrichesvariant DNA sequences and redefines the sensitivity of genetic testing.Nat Med 2008; 14:579-84; Milbury C A, Li J, Makrigiorgos G M.Ice-COLD-PCR enables rapid amplification and robust enrichment forlow-abundance unknown DNA mutations. Nucleic Acids Res; 39:e2). However,the enrichment that can be obtained via such PCR-based methods has alimit, since after several cycles of synthesis, the polymeraseunavoidably introduces mis-incorporations (PCR errors) that aresubsequently scored as mutations. Repeated amplifications can alsointroduce mis-priming which results in the amplification of unwantednon-target sequences. Furthermore, there are powerful genetic analysismethods currently emerging (‘third generation sequencing’ Nanopore,Pac-Bio systems) that may obviate the use of PCR altogether. Therefore,mutation enrichment methods that reduce the amount of PCR performed, orthat can operate without PCR, or in conjunction with PCR if so required,are important.

The present disclosure is based, at least in part, on the noveldevelopment of a technique, Nuclease-assisted Mutation Enrichment (NaME)that results in the preferential cleavage of non-variant/wild-type DNAor RNA, thereby allowing for subsequent selective enrichment of variant(mutant) target sequences. Thus, NaME can be used before, during, orafter an amplification step, such as PCR, or without any amplification,depending on the application. Subsequently, the mutation-enrichedsequences can be screened via any currently available method foridentifying mutations, including Sanger Sequencing, high resolutionmelting (HRM), SSCP, next generation massively parallel sequencing, andMALDI-TOF for known mutations; and Single Molecule Sequencing- or thirdgeneration sequencing for high-throughput sequencing of low-levelmutations. NaME can also be applied to detect low levels ofun-methylated alleles (Methylation-Sensitive Nuclease-assistedminor-allele Enrichment or MS-NaME) in a background of methylatedalleles (or vice-versa).

The methods described herein greatly improve the current detectionlimits of mutation/methylation detection technologies, thereby enhancingreliability of patient-specific mutation screening, for example, inheterogeneous tumor samples or in circulating DNA. The methods describedherein also enable high multiplexity of targets (i.e., enable thesimultaneous screening of a panel of DNA regions), thus enablinghigh-throughput methods to be used for somatic mutation detection, (forexample, massively parallel sequencing, MPS). NaME is particularlyuseful in the field of circulating biomarkers for cancer applications,pre-natal diagnostic applications, and infectious disease applications.

‘Wild-type target sequence’ refers to a nucleic acid that is moreprevalent in a nucleic acid sample than a corresponding target sequence(e.g., same region of gene but different nucleic acid sequence). Thewild-type sequence makes-up over 50% of the total wild-typesequence+mutant target sequence in a sample. The wild-type sequence canbe expressed at the RNA and/or DNA level 10×, 15×, 20×, 25×, 30×, 35×,40×, 45×, 50×, 60×, 70×, 80×, 100×, 150×, 200× or more than the targetsequence. For example, a sample (e.g., blood sample) may containnumerous normal cells and few cancerous cells. The normal cells containwild-type alleles (non-mutant) sequences, while the small number ofcancerous cells contain target sequences. As used herein, a ‘wild-typestrand’ refers to a single nucleic acid strand of a wild-type sequence.The term ‘wild-type’ typically refers to the most common polynucleotidesequence or allele for a certain gene in a population. Generally, thewild-type allele will be obtained from normal cells.

The wild-type sequence is about 13-2000 nucleotides long. In someembodiments, the wild-type sequence is 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 900 or more nucleotides long. Wild-typesequences will share at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the correspondingtarget sequence, but will differ by at least one nucleotide from thetarget sequence. In some embodiments, the at least one nucleotide is amethylated cytosine. In some embodiments, the at least one nucleotide isan unmethylated cytosine. Wild-type sequences according to the inventioncan be amplified by PCR with the same pair of primers as that used forthe mutant sequence.

A ‘target nucleic acid’ or ‘target sequence’, used interchangeablyherein, refers to a nucleic acid that is less prevalent in a nucleicacid sample than a corresponding wild-type sequence. The target sequencemakes-up less than 50% of the total amount of wild-type sequence+targetsequence in a sample. Preferably the target sequence is expressed at theRNA and/or DNA level 1:10, 1:15, 1:20, 1:25×, 1:30, 1:35, 1:40, 1:45,1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200× or less than thewild-type sequence. In some embodiments, the target sequence is a mutantallele. For example, a sample (e.g., blood sample) may contain numerousnormal cells and few cancerous cells. The normal cells contain wild-type(i.e., non-mutant) sequences, while the small number of cancerous cellscontain target mutant sequences. In some embodiments, the targetsequence is repeat sequences that occur at large numbers in human genome(including but not limited to ALU elements, LINE elements, SINEelements, di-nucleotide repeats, tri-nucleotide repeats). Altered repeatsequences occur often in diseased states and application of the presentinvention in detecting alterations in repeat sequences is of interest.In some embodiments, the methods described herein are directed todetecting fetal DNA in a nucleic acid sample obtained from a mother. Inthis embodiment, the fetal DNA is the target sequence while the moreprevalent mother DNA is the wild-type sequence. In some embodiments, thetarget sequence is a methylated allele. In some embodiments, the targetsequence is an unmethylated allele. As used herein, a “target strand”refers to a single nucleic acid strand of a target sequence.

In some embodiments, the target sequence is 13-2000 nucleotides long. Insome embodiments, the target sequence is 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 900 or more nucleotides long. Targetsequences share at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more homology to the correspondingwild-type sequence, but differs by at least one nucleotide from thewild-type sequence. In some embodiments, the at least one nucleotide isa methylated cytosine. In some embodiments, the at least one nucleotideis an unmethylated cytosine. Target sequences according to the inventioncan be amplified via PCR with the same pair of primers as those used forthe wild-type sequence.

‘Target mutant sequence’ or ‘mutant target sequence’ refers to a nucleicacid that is less prevalent in a nucleic acid sample than acorresponding wild-type sequence. The target mutant sequence typicallymakes-up less than 50% of the total amount of wild-type sequence+mutantsequence in a sample. The target mutant sequence may be expressed at theRNA and/or DNA level 1:10, 1:15, 1:20, 1:25×, 1:30, 1:35, 1:40, 1:45,1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200× or less than thewild-type sequence. For example, a sample (e.g., blood sample) maycontain numerous normal cells and few cancerous cells. The normal cellscontain wild-type (non-mutant) alleles, while the small number ofcancerous cells contain target mutant sequences. In some embodiments,the invention is directed to detecting fetal DNA in a nucleic acidsample obtained from a mother. In this embodiment, the target mutantsequence is the fetal DNA while the more prevalent mother DNA is thewild-type sequence. As used herein, a target mutant sequence is meant toinclude fetal DNA obtained from a pregnant mother. In some embodiments,the present disclosure is directed to detecting one or more methylatedalleles in the presence of a large excess of unmethylated alleles, orvice versa in epigenetic analysis.

The target mutant sequence is about 13-2000 nucleotides long. In someembodiments, the target mutant sequence is 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, 450, 500, 600, 700, 800, 900 or more nucleotides long. Targetmutant sequences share at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the correspondingwild-type sequence, but differs by at least one nucleotide from thewild-type sequence. Mutant target sequences according to the inventioncan be amplified via PCR with the same pair of primers as those used forthe wild-type sequence.

The term ‘mutant’ refers to a nucleotide change (i.e., a single ormultiple nucleotide substitution, deletion, insertion, or methylation,or alteration in the number of poly-nucleotide repeats) in a nucleicacid sequence. A nucleic acid which bears a mutation has a nucleic acidsequence (mutant allele) that is different in sequence from that of thecorresponding wild-type sequence. The methods described herein areespecially useful in preferentially cleaving wild-type sequences,thereby allowing for selective enrichment of several or numerous mutanttarget sequences simultaneously. The mutant alleles can contain between1 and 500 nucleotide sequence changes. A mutant allele may have 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nucleotidesequence changes compared to a corresponding wild-type allele.Typically, a mutant allele will contain between 1 and 10 nucleotidesequence changes, and more typically between 1 and 5 nucleotide sequencechanges. The mutant allele will have 50%, 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the wild-typeallele. Generally, the mutant allele will be obtained from diseasedtissues or cells and is associated with a disease state.

As used herein, a ‘region of interest’ is a sequence that will beinterrogated for variations such as clinically relevant mutations, andmethylation/unmethylation patterns.

‘Enriching a mutant target sequence’ refers to increasing the amount ofa mutant target sequence and/or increasing the ratio of mutant targetsequence relative to the corresponding wild-type sequence in a sample.For example, where the ratio of mutant sequence to wild-type sequence isinitially 5% to 95% in a sample, the mutant sequence may bepreferentially amplified in an amplification reaction so as to produce aratio of 70% mutant sequence to 30% wild-type sequence. Thus, there is a14-fold enrichment of the mutant sequence relative to the wild-typesequence in this hypothetical example. Generally, enrichment of a mutanttarget sequence results in a 2× to 200× increase in the mutant targetsequence relative to the wild-type sequence prior to enrichment. Theenrichment of the mutant target sequence is at least a 2×, 3×, 4×, 5×,6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×, 30×, 35×, 40×, 45×, 50×, 60×, 80×,90×100×, 150×, 200× or more fold enrichment. Enrichment of a mutanttarget sequence results in a sample having 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 95% or more, mutant targetsequence compared to wild-type sequence (e.g., 10% mutant targetsequence:90% wild-type sequence to 95% mutant target sequence:5%wild-type sequence).

‘Allele’ refers to alternative forms of a gene, portion thereof ornon-coding region of DNA that occupy the same locus or position onhomologous chromosomes that have at least one difference in thenucleotide sequence. The term allele can be used to describe DNA fromany organism including but not limited to bacteria, viruses, fungi,protozoa, molds, yeasts, plants, humans, non-humans, animals, andarchaebacteria. The alleles may be found in a single cell (e.g., twoalleles, one inherited from the father and one from the mother) orwithin a population of cells (e.g., a wild-type allele from normaltissue and a somatic mutant allele from diseased tissue).

An allele can be 13-2000 nucleotides long. In one embodiment the alleleis 20, 30, 40, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or morenucleotides long. Alleles will generally share 50%, 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology toeach other. Alleles according to the invention can be amplified by PCRwith the same pair of primers.

In one embodiment, the methods described herein are used to enrich apolymorphism. Any given gene may have none, one, or many allelic forms(polymorphism). Common mutational changes which give rise to alleles maybe the result of natural or artificial (e.g., chemical carcinogens)deletions, additions, or substitutions of nucleotides. Each of thesetypes of changes may occur alone, or in combination with the others, oneor more times in a given sequence.

As used herein the term ‘melting temperature’ or ‘I'm’ refers to thetemperature at which a polynucleotide dissociates from its complementarysequence. Generally, the Tm may be defined as the temperature at whichone-half of the Watson-Crick base pairs in a duplex nucleic acidmolecule are broken or dissociated (i.e., are ‘melted’) while the otherhalf of the Watson-Crick base pairs remain intact in a double strandedconformation. In other words, the Tm is defined as the temperature atwhich 50% of the nucleotides of two complementary sequences are annealed(double strands) and 50% of the nucleotides are denatured (singlestrands). Tm, therefore, defines a midpoint in the transition fromdouble-stranded to single-stranded nucleic acid molecules (or,conversely, in the transition from single-stranded to double-strandednucleic acid molecules).

The Tm can be estimated by a number of methods, for example by anearest-neighbor calculation as per Wetmur 1991 (Wetmur, J. G. 1991. DNAprobes: applications of the principles of nucleic acid hybridization.Crit. Rev. Biochem. Mol. Biol., 26: 227-259, hereby incorporated byreference) and by commercial programs including Oligo™ Primer Design andprograms available on the internet. Alternatively, the Tm can bedetermined though actual experimentation. For example, double-strandedDNA binding or intercalating dyes, such as ethidium bromide orSYBR-green (Molecular Probes) can be used in a melting curve assay todetermine the actual Tm of the nucleic acid. Additional methods fordetermining the Tm of a nucleic acid are well known in the art anddescribed herein.

As used herein, ‘reaction mixture’ refers to a mixture of constituentsincluding but not limited to a nucleic acid sample comprising adouble-stranded wild-type nucleic acid and a double-stranded targetnucleic acid suspected of containing a mutation, and a pair ofoligonucleotide probes that are complementary to top and bottom strandsof the wild-type nucleic acid. The reaction mixture can also includereagents, such as, but not limited to, salt(s), buffer(s), and enzyme(s)such as double strand-specific nuclease (DSN), exonuclease, andpolymerase.

As used herein, a nucleic acid sample refers to any substance containingor presumed to contain a nucleic acid of interest (target and wild-typesequences), or which is itself a nucleic acid containing or presumed tocontain a target nucleic acid of interest. The term “nucleic acidsample” thus includes a sample of nucleic acid (genomic DNA, cDNA, RNA),cell, organism, tissue, or fluid, including but not limited to, forexample, plasma, serum, spinal fluid, lymph fluid, synovial fluid,urine, tears, stool, external secretions of the skin, respiratory,intestinal and genitourinary tracts, saliva, blood cells, tumors,organs, tissue, samples of in vitro cell culture constituents, naturalisolates (such as drinking water, seawater, solid materials), microbialspecimens, and objects or specimens that have been “marked” with nucleicacid tracer molecules. The nucleic acid sample may be obtained frommammals, viruses, bacteria or plants. In some embodiments, the nucleicacid sample is DNA circulating in plasma, urine or other bodily fluids.

As used herein “oligonucleotide probes” refer to molecules comprisingtwo or more deoxyribonucleotides or ribonucleotides. The methodsdescribed herein utilize a pair of oligonucleotide probes, one of whichis complementary to the wild-type nucleic acid top strand and the otheris complementary to the wild-type nucleic acid bottom strand. By“complementary” it is meant that the probes hybridize without anymismatches to the sequences in the top and bottom stands of thewild-type nucleic acid. The oligonucleotide probes are non-identical,i.e., the sequences of the two probes are different from each other. Insome embodiments, the probes do not overlap each other, i.e., they donot bind to each other. At least one of the probes overlaps a sequenceon the target nucleic acid containing the suspected mutation, i.e., theprobe hybridizes to the sequence on the target nucleic acid containingthe suspected mutation with at least one mismatch thereby forming a“partially complementary” target mutant-probe duplex.

In some embodiments, one of the probes overlaps a sequence on the topstrand of the target nucleic acid containing the mutation, while theother probe overlaps a sequence on the bottom strand of the targetnucleic acid containing the mutation. Thus, the probes hybridizerespectively to the top and bottom sequences on the target nucleic acidcontaining the suspected mutation with at least one mismatch. In suchembodiments, the probes partially overlap each other. However, they donot bind substantially to each other.

The oligonucleotide probes may be anywhere between 5 and 100 bases long.In some embodiments, the probes are 5, 10, 20, 30, 40, 50, 60, 70, 80,90, or 100 bases long. In some embodiments, the probes are in a molarexcess of 100-fold- to 1 billion-fold compared to the wild-type andtarget nucleic acids (e.g., 100-fold, 500-fold, 1000-fold, 10,000-fold,50,000-fold, 100,000-fold, 500,000-fold, 1 million-fold, 500-millionfold, 100 million-fold, 1 billion-fold in excess as compared to thewild-type and target nucleic acids). In some embodiments the probes arein a molar concentration of 1 μM, 10 μM, 50 μM, 100 μM, 200 μM, 300 μM,400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, or 1,000 μM in thereaction.

By “selectively cleaved” or “preferentially cleaved” is meant that thesubject methods preferentially cut, i.e., cleave, deoxyribonucleic acidmolecules present in perfectly matched double-stranded nucleic acids,e.g., DNA-DNA duplexes and DNA-RNA duplexes. Perfectly matcheddouble-stranded nucleic acids are hybrid structures betweencomplementary strands where no mismatches are present, as compared topartially complementary nucleic acid duplexes of the same length. Thus,in the methods described herein complementary DNA containing duplexnucleic acids (i.e., without any mismatches) are cleaved to a muchgreater extent than partially complementary nucleic acid duplexes (i.e.,with one or more mismatches), non-DNA containing nucleic acid duplexesand/or single-stranded nucleic acids. In other words, the subjectmethods are able to cleave or cut perfectly matched nucleic acidsduplexes in a sample at a much greater rate than other nucleic acidmolecules that may be present in the sample being treated, where therate of perfectly matched nucleic acids duplex cleavage is typically atleast 5 fold, at least 10 fold, at least 50 fold, or at least 100 foldgreater than the rate of cleavage of other nucleic acids that may bepresent in the sample being treated.

As used herein, “primers” refers to oligonucleotides that anneal toopposite strands of a mutant target and wild-type sequence so as to forman amplification product during a PCR reaction.

NaME on Double Stranded DNA

Accordingly, some aspects of the disclosure provide methods forpreparing a target mutant nucleic acid for subsequent enrichmentrelative to a wild-type nucleic acid. The subsequent enrichment can beachieved by applying amplification conditions to the produced reactionmixture.

In some embodiments, the method comprises subjecting a nucleic acidsample comprising a double-stranded wild-type nucleic acid and adouble-stranded target nucleic acid suspected of containing a mutationto a condition that destabilizes the double stranded wild-type andtarget mutant nucleic acids; contacting the destabilized double strandedwild-type and target mutant nucleic acids with a pair of oligonucleotideprobes, one of which is complementary to the wild-type nucleic acid topstrand and the other is complementary to the wild-type nucleic acidbottom strand, to permit hybridization of the probes to theircorresponding sequences on the wild-type and target mutant nucleic acidsthereby forming complementary wild-type-probe duplexes on top and bottomstrands, and partially complementary target mutant-probe duplexes,wherein at least one of the probes overlaps a sequence on the targetnucleic acid containing the suspected mutation; and exposing thecomplementary wild-type-probe duplexes and the partially complementarytarget mutant-probe duplexes to a double strand-specific nuclease (DSN),wherein the DSN cleaves the complementary wild-type-probe duplexes butnot the partially complementary target mutant-probe duplexes.

In some embodiments, the method comprises exposing a nucleic acid samplecomprising a double-stranded wild-type nucleic acid and adouble-stranded target nucleic acid suspected of containing a mutationto a double strand-specific nuclease (DSN) and a pair of oligonucleotideprobes, one of which is complementary to the wild-type nucleic acid topstrand and the other is complementary to the wild-type nucleic acidbottom strand, to create a reaction mixture, wherein at least one of theprobes overlaps a sequence on the target nucleic acid containing thesuspected mutation; and subjecting the reaction mixture to a conditionthat destabilizes the double stranded wild-type and target mutantnucleic acids to permit hybridization of the probes to theircorresponding sequences on the wild-type and target mutant nucleic acidsthereby forming complementary wild-type-probe duplexes on top and bottomstrands, and partially complementary target mutant-probe duplexes,wherein the DSN cleaves the complementary wild-type-probe duplexes butnot the partially complementary target mutant-probe duplexes.

NaME utilizes nucleases (DNases) that show a strong preference forcleaving double stranded DNA versus single stranded DNA or RNA. DNasesthat can be used in the methods described herein include, but are notlimited to, native shrimp dsDNase, recombinant shrimp dsDNase, King crabnuclease (DSN) and bovine DNase I. NaME takes advantage of the DSNproperties to cleave specific sequences from both top and bottom DNAstrands of wild-type DNA as shown on FIG. 1A. In contrast,mutation-containing DNA is not cleaved or cleaved to a significantlyless extent than wild-type DNA. Hence, a subsequent PCR reaction afterDSN digestion amplifies preferentially the mutant alleles that remainsubstantially intact and leads to enrichment of mutant versus wild-typealleles.

For the purposes of the present disclosure, the term “double-strandspecific nuclease” or “DSN” includes DNA/RNA guided enzymes which havepreferential activity on double-stranded DNA, as compared to singlestranded DNA. Examples of such enzymes that can be employed inconjunction with NaME include the RNA-guided Cas9 enzymes (Gu et al,Depletion of Abundant Sequences by Hybridization (DASH): Using Cas9 toremove unwanted high-abundance species in sequencing libraries andmolecular counting applications Genome Biology 2016; 17, 41), or theArgonaute DNA-guided enzymes (Gao et al, DNA-guided genome editing usingthe Natronobacterium gregoryi Argonaute, Nature Biotechnology May 2016advanced online publication). These DNA/RNA guided enzymes digest DNAwith high preference when the probe (‘guide oligonucleotide’) is fullymatched to the target DNA, and less so when there is a mismatch. Byemploying probes targeting both top and bottom DNA strands in anoverlapping fashion as described in the present invention, NAME can beapplied with DNA/RNA guided enzymes, in the same manner as when usingother DSN nucleases described herein.

During NaME, (FIG. 1A) DSN and a pair of oligonucleotide probes thatmatch the top and bottom strands of the wild-type nucleic acid ofinterest are added to (i.e., exposed or contacted with) a nucleic acidsample comprising double-stranded wild-type nucleic acid anddouble-stranded target nucleic acid suspected of containing a mutationto create a reaction mixture. The nucleic acid sample is exposed to theDSN and the oligonucleotide probes at a low temperature at which the DSNis inactive (e.g., 4° C.). At least one of the oligonucleotide probesoverlaps sequences on the target nucleic acid that are suspected ofcontaining clinically important mutations (e.g., KRAS codon 12/13sequences; p53 sequences; tri-nucleotide repeat sequences; etc.). Thesecond oligonucleotide binds the opposite target nucleic acid strandfrom the first oligonucleotide probe and can have similar length as thefirst oligonucleotide. In some embodiments, this second probe isdesigned to match a sequence on the target nucleic acid that normallydoes not contain mutations. In some embodiments, the probes are in amolar excess of 100-fold, 500-fold, 1000-fold, 10,000-fold, 50,000-fold,100,000-fold, 500,000-fold, 1 million-fold, 500 million-fold, 100million-fold, 1 billion-fold compared to the wild-type and targetnucleic acids.

The reaction mixture is then subjected to a condition that destabilizesthe double stranded wild-type and mutant nucleic acids to permithybridization of the probes to their corresponding sequences on thewild-type and mutant nucleic acids thereby forming complementarywild-type-probe duplexes on top and bottom strands, and partiallycomplementary mutant-probe duplexes. By “destabilizing” it is meant thatthe double stranded wild-type and target mutant nucleic acids denatureto such an extent so as to allow the probes to hybridize to theircorresponding sequences, but the wild-type and target mutant nucleicacids do not denature completely. A condition that destabilizes thedouble stranded wild-type and mutant nucleic acids to permithybridization of the probes to their corresponding sequences on thewild-type and mutant nucleic acids include addition of an organicsolvent such as, but not limited to DMSO, betaine or formamide and/or anincrease in temperature combined with a thermostable DSN. The increasein temperature is such that it enables specific probe hybridization toits corresponding sequence. The temperature of the reaction mixture israised to a temperature that destabilizes the double stranded structure(e.g., 65° C.-80° C. including 65° C., 70° C., 75° C., 80° C.) but doesnot denature it completely. This destabilizing temperature is typicallyabout 10-20° C. below the melting temperature (Tm) of the nucleic acidsequence. At this temperature, the oligonucleotide probes invade andbind to their corresponding sequences on the wild-type and mutantnucleic acids. The probes fully match the sequences on the wild-typenucleic acid and can, thus, form complementary wild-type-probe duplexes(i.e., with no mismatches).

If a suspected mutation is present on the target nucleic acid, thebinding between the probe and the target nucleic acid is inefficient andresults in partially complementary mutant-probe duplexes (i.e., withmismatches). The complementary wild-type-probe duplexes are recognizedand cleaved by the DSN enzyme. In contrast, the partially complementarymutant-probe duplexes remain substantially intact.

In some embodiments, one of the oligonucleotide probes overlaps asequence on the target nucleic acid that is suspected of containing amutation while the second probe is designed to match a sequence at adifferent position on the target nucleic acid that normally does notcontain mutations (FIG. 1A). Hence, the approach shown on FIG. 1Atypically leads to cleavage of both strands for wild-type nucleic acidwhile only a single DNA strand of the mutant nucleic acid is cleaved.

In some embodiments, the methods described herein are performed by firstdestabilizing the double-stranded wild-type nucleic acid and thedouble-stranded target nucleic acid suspected of containing a mutation.The destabilized wild-type nucleic acid and the target mutant nucleicacid are then contacted with the oligonucleotide probes to permithybridization of the probes to their corresponding sequences on thewild-type and target mutant nucleic acids thereby forming complementarywild-type-probe duplexes on top and bottom strands, and partiallycomplementary target mutant-probe duplexes. By “contacting” it is meantthat the probes are added to the nucleic acids and the components aremixed, or the nucleic acids are added to the probes and the componentsare mixed. The duplexes are then exposed to DSN which preferentiallycuts the complementary wild-type-probe duplexes but not the partiallycomplementary target mutant-probe duplexes.

Some aspects of the disclosure provide methods for preparing a targetmutant nucleic acid for subsequent enrichment relative to a wild-typenucleic acid comprising exposing a nucleic acid sample comprising adouble-stranded wild-type nucleic acid and a double-stranded targetnucleic acid suspected of containing a mutation to a pair ofoligonucleotide probes, one of which is complementary to the wild-typenucleic acid top strand and the other is complementary to the wild-typenucleic acid bottom strand, to create a reaction mixture, wherein atleast one of the probes overlaps a sequence on the target nucleic acidcontaining the suspected mutation; subjecting the reaction mixture to adenaturing temperature to permit denaturation of the wild-type nucleicacid and the target mutant nucleic acid; reducing the temperature of thereaction mixture to permit formation of complementary wild-type-probeduplexes on top and bottom strands and partially complementary targetmutant-probe duplexes; and exposing the reaction mixture to a doublestrand-specific nuclease (DSN), wherein the DSN cleaves thecomplementary wild-type-probe duplexes but not the partiallycomplementary target mutant-probe duplexes.

In these methods, the double stranded wild-type and target nucleic acidsin the presence of the two probes are first denatured by subjecting thereaction mixture to a denaturing temperature, while also optionallyincluding organic solvents like DMSO, betaine or formamide. Thedenaturing temperature should be sufficiently high so as to allow thefull denaturation of the wild-type and target nucleic acids (e.g., 75°C., 80° C., 85° C., 90° C., or 95° C.). In some embodiments, thedenaturing temperature is about 1° C. to 30° C. above the Tm of thewild-type and nucleic acid sequence (e.g., 1° C., 5° C., 10° C., 15° C.,20° C., 25° C., 30° C. above the Tm of the wild-type and nucleic acidsequence).

Next the temperature of the reaction mixture is decreased allowing thewild-type and target nucleic acids to hybridize with the oligonucleotideprobes to form complementary wild-type-probe duplexes on top and bottomstrands (i.e., with no mismatches) and partially complementarymutant-probe duplexes (i.e., with mis-matches). In some embodiments,this hybridization temperature is 40° C., 45° C., 50° C., 55° C., 60°C., 65° C., 70° C., or 75° C.). At this hybridization temperature, sincethe two probes are in high excess relative to the target nucleic acid,they bind first to their respective targets, i.e., while the two parentstrands of the wild-type and target nucleic acids have not yetre-associated and remain substantially single-stranded. In someembodiments, the probes are in a molar excess of 100-fold, 500-fold,1000-fold, 10,000-fold, 50,000-fold, 100,000-fold, 500,000-fold, 1million-fold, 500 million-fold, 100 million-fold, 1 billion-foldcompared to the wild-type and target nucleic acids.

In this method, DSN is, optionally, not added from the beginning inorder to avoid partial or total inactivation of the DSN at thedenaturing temperature. DSN is added once the temperature is reduced toallow formation of complementary wild-type-probe duplexes on top andbottom strands and partially complementary mutant-probe duplexes. DSNthen preferentially degrades the complementary wild-type-probe duplexes,while the partially complementary mutant-probe duplexes remainsubstantially intact. The DSN activity can then be stopped, for example,by heating the sample to 95° C. for 1-10 min to inactivate the DSN. Asubsequent PCR reaction amplifies preferentially the mutated allelesthat remain substantially intact, while the DSN-digested wild-typealleles do not amplify. FIG. 1B demonstrates quantification of thefractional mutation abundance following this PCR reaction. It can beseen that in the absence of DSN and/or both probes the mutationabundance is low (4-6%) while in the presence of DSN and both probes theresulting mutation abundance is 37-38%, i.e., demonstrating theenrichment of the mutated alleles using NaME.

In some embodiments, the subsequent PCR reaction amplifies the probesrather than the hybridized nucleic acid. In this approach, apurification step is applied following probe binding to top-and-bottomDNA target strands, either before or after DSN cleavage, to removeexcess unbound probes (e.g., using a DNA affinity column, such asQIAquick® PCR purification kit commercially available from QIAGEN). Thenfollowing DSN cleavage the uncut probes are amplified (instead ofamplifying the target DNA) and identified/quantified. Since probes thatbind WT DNA will have been selectively digested by DSN, the presence ofany given probe after amplification indicates a mutation under theregion covered by this probe.

NaME Using Overlapping Probes

In some embodiments of the methods described herein, the probes areconstructed such that one of the probes overlaps a sequence on the topstrand of the target nucleic acid containing the mutation, while theother probe overlaps a sequence on the bottom strand of the targetnucleic acid containing the mutation of interest and the two probespartially overlap each other (FIGS. 2 and 3 ). The two probes overlaponly partially, so that they do not bind substantially to each other insolution at the temperatures used for NaME during DSN digestion (e.g.,65° C.-70° C.). Accordingly, it is important during probe hybridizationto their corresponding sequences to retain a temperature low enough forprobe binding to the template, but high enough so that it does not allowsubstantial probe-to-probe binding. This approach increases thespecificity of the process for mutated sequences, and the mutationenrichment becomes much more pronounced than when using only onemutation-specific probe with a second probe which matches the wild-typenucleic acid (FIG. 1A).

In some embodiments, a thermostable DSN, such as but not limited to,king crab nuclease is used in the methods described herein. In someembodiments, a non-thermostable DSN, such as but not limited to, nativeshrimp dsDNase, recombinant shrimp dsDNase, and bovine DNase I is usedin the methods described herein. If a non-thermostable DSN is usedduring NaME, then the overlap between the probes designed must be suchthat at the temperature used for probe-nucleic acid duplex formation(e.g., 37-45° C. there is minimal binding of the probes to each other,while they still bind specifically to genomic DNA targets). One way toreduce the Tm of the probes to match the optimal temperature of thenuclease used is to add organic solvents (DMSO, formamide) that lowerthe Tm of the probes. For example, instead of probes with a Tm of 65° C.matching the optimal temperature of thermostable DSN enzyme, one may useshrimp nuclease in the presence of 10% DMSO which reduces the probe Tmas well as the Tm of probe-probe overlap regions.

For the purposes of the present disclosure, the term “double-strandspecific nuclease” or “DSN” includes DNA/RNA guided enzymes which havepreferential activity on double-stranded DNA, as opposed to singlestranded DNA. Examples of such enzymes that can be employed inconjunction with NaME include the RNA-guided Cas9 enzymes (Gu et al,Depletion of Abundant Sequences by Hybridization (DASH): Using Cas9 toremove unwanted high-abundance species in sequencing libraries andmolecular counting applications Genome Biology 2016; 17, 41), or theArgonaute DNA-guided enzymes (Gao et al, DNA-guided genome editing usingthe Natronobacterium gregoryi Argonaute, Nature Biotechnology May 2016advanced online publication). These DNA/RNA guided enzymes digest DNAwith high preference when the probe (‘guide oligonucleotide’) is fullymatched to the target DNA, and less so when there is a mismatch. Byemploying probes targeting both top and bottom DNA strands in anoverlapping fashion as described in the present invention, NAME can beapplied with DNA/RNA guided enzymes, in the same manner as when usingother DSN nucleases described herein.

In some embodiments, prior to implementing the methods described herein,the nucleic acid sample is subjected to an amplification condition. Insome embodiments, the methods described herein further compriseenriching the target mutant nucleic acid relative to the wild-typenucleic acid by subjecting the reaction mixture containing cleavedwild-type-probe duplexes and uncleaved target mutant nucleic acids to anamplification condition thereby enriching the uncleaved target mutantnucleic acid relative to the cleaved wild-type nucleic acid. Any knownamplification condition can be used. In some embodiments, theamplification condition is selected from the group consisting of: PCR,ligation mediated PCR using common ligated adaptors, multiplex PCR,using multiple pairs of primers, PCR of repeat elements using primersspecific for ALU, LINE 1, poly-nucleotide repeats, micro-satellites andother repeat elements spread over the genome, arbitrarily-primed PCR(AP-PCR) and isothermal amplification (such as but not limited todisplacement amplification based on phi-29 based; or Loop Mediated LAMPamplification; or any other isothermal mode of amplification). Thewild-type nucleic acid will not amplify during this amplification stepsince it was cleaved selectively by DSN. The mutation-enriched amplifiedproduct can then be analyzed for mutations using any available methodsuch as MALDI-TOF, HR-Melting, Di-deoxy-sequencing, Single-moleculesequencing, massively parallel sequencing (MPS), pyrosequencing, singlestrand conformational polymorphism SSCP, restriction fragment lengthpolymorphism RFLP, denaturing high precision liquid chromatographydHPLC, chemical cleavage of mismatches CCM, capillary electrophoresis,digital PCR and quantitative-PCR.

The probes used in the methods described herein preferably contain a3′-block to polymerase extension, so that if the NaME-reaction productis subsequently amplified there is no interference of the probes withthe amplification reaction. A 3′-polymerase block can comprise a simplephosphate; or abasic site; or any other modification that preventspolymerase synthesis past the block. In addition, for addeddiscrimination of wild-type versus mutant sequences during NaME, in someembodiments, each probe comprises a locked nucleic acid (LNA), peptidenucleic acid (PNA), xeno nucleic acid (XNA), nucleic acid with any knownnatural or modified base such as dITP or 2,6-diaminopurine dATP or RNAthat increases the destabilization caused by a mutation-induced mismatchbetween the oligonucleotide probe and its target nucleic acid.

In some cases, part of the probes used in the methods provided hereincan comprise one or more random nucleotides, so that the probe can bedirected against a plurality of DNA targets. For example, a probe caninclude a core region of one or more nucleotides which are complementaryto the wild-type nucleic acid sequence in a region of interest. e.g., asuspected mutation site. Any one or more of the remaining nucleotides inthe probe may be selected randomly from any or all possible nucleotides.The probe containing the core region plus one or more random nucleotidescan form a duplex with a fully complementary wild-type nucleic acidsequence which also contains the core region. A cleavage enzyme, e.g.,DSN, can be used to cleave the complementary wild-type probe duplexes,but not the partially complementary target mutant-probe duplexes.

In some embodiments, the methods described herein are used to preparetwo or more different target mutant nucleic acids for subsequentenrichment relative to corresponding wild-type nucleic acids. In suchembodiments, one or more additional pairs of probes directed to thedifferent wild-type nucleic acids are used. For each pair of probes, oneof the probes is complementary to the wild-type nucleic acid top strand,while the other is complementary to the wild-type nucleic acid bottomstrand.

In all embodiments described above, it is also understood that theconcentration of probes for the top and bottom DNA strands does notnecessarily need be the same. Thus, one may combine a high concentrationof probe for the bottom strand and a low concentration of probe for thetop strand, or vice versa. Probe concentrations can also be differentfor each DNA target when many targets are simultaneously enriched.Optimized concentrations depending on sequence context, local sequenceTm and mutation being targeted can be applied. Furthermore, a subsequentPCR amplification reaction following application of DSN can utilizeequal amounts of primers or different amounts of primers for each DNAstrand (asymmetric PCR, or Linear After The Exponential, L.A.T.E PCR).

NaME Applied Directly on Genomic DNA

In some embodiments, the methods described herein are performed directlyon genomic DNA. The genomic DNA can optionally be fragmented prior toapplication of NaME (FIG. 4 ). In this approach, NaME is applied by (a)fragmenting the genomic DNA using enzymatic or physical means; (b)adding overlapping (or non-overlapping) probes that address both top andbottom DNA strands and optionally denaturing both the wild-type andtarget nucleic acids (for example, at 95° C.); (c) reducing thetemperature to, for example, 60-70° C. to enable probes to find theirrespective targets prior to substantial renaturation of the parent DNAstrands, and keeping the temperature high enough to minimizeprobe-to-probe interactions; (d) adding DSN to selectively cleave one ormore (multiple) complementary wild-type-probe duplexes DNA targets whileleaving the partially complementary target mutant-probe duplexessubstantially intact. The resulting reaction mixture with cleavedwild-type-probe duplexes and uncleaved target mutant nucleic acids canbe amplified using methods known in the art, such as but not limited to,PCR, COLD-PCR, ligation-mediated PCR or COLD-PCR using common ligatedadaptors, multiplex-PCR or isothermal amplification (such as phi-29based; or LAMP-based; or any other isothermal mode of amplification)thereby enriching the target mutant nucleic acid as compared to thewild-type. This amplified product can be examined for mutations usingany available method, such as but not limited to, MALDI-TOF, HR-Melting,Di-deoxy-sequencing, Single-molecule sequencing, massively parallelsequencing (MPS), pyrosequencing, SSCP, RFLP, dHPLC, CCM, digital PCRand quantitative-PCR (the wild-type nucleic acid will not amplify duringthis amplification step since it was selectively cleaved by DSN).

FIG. 5 depicts the enrichment of an original ˜1% mutation to an 83%mutation and a 0.5% mutation to 14% mutation, following application ofNaME directly to genomic DNA. FIG. 6 shows a duplex application of NaMEon two mutated targets simultaneously, in KRAS and TP53 genes. FIG. 7Ashows application of NaME using two overlapping probes covering 3different mutations in codons 12 and 13 of Kras and indicating that anymutation under the probes will be enriched during NaME. And FIG. 7Bdemonstrates simultaneous enrichment of 11 different targets when NaMEis applied directly from genomic DNA. For each target, a separate pairof overlapping probes was designed, and all probes are included in asingle reaction during NaME.

In some embodiments the probes used are directed against polynucleotiderepeats that are widespread around the genome, so that multiple targetsare addressed via NaME simultaneously, using a single pair of probes.For example, if the target is a poly-A-containing sequence, twooverlapping probes are used, one for bottom strand containing poly-T andone for top strand containing poly-A in this case. To increase thelength of the probe, one may also add an optional number of inosinesthat can generically bind to neighboring bases.

Combination of NaME with Massively Parallel Sequencing (MPS).

In some embodiments, the methods described herein may be used inconjunction with massively parallel sequencing (MPS). MPS is currentlythe most advanced approach for mutation identification. Sample(‘library’) preparation for MPS is a very important step prior toapplying genome-wide or exome-wide sequencing or targeted re-sequencing.NaME provides a unique advantage for sample preparation prior to MPS, asit can enrich predictably numerous targets for mutations, therebyenabling MPS to identify easily mutations that are originally at verylow abundance, without requiring an excessive number of sequence reads.This enables cost reduction and increased sensitivity and simplicity.FIG. 8 provides an example of NaME—enhanced sample preparation processprior to MPS. In this approach, multiplexed NaME (using overlappingprobes against numerous gene mutations simultaneously as described inprevious sections) is applied to the original starting material in orderto cleave selectively wild-type nucleic acid on all targets of interestsimultaneously. The sample is then amplified to enrich preferentiallythe mutated DNA targets. Finally, the resulting mutation-enriched DNA isused for routine library construction prior to MPS.

In some embodiments, multiplexed application of NaME on numerous targetsof interest can be applied directly from denatured genomic DNA asdescribed in FIG. 4 . Following this, a multiplexed PCR can be appliedusing primers addressing the DNA targets of interest (for example, butnot limited to, the primers used in the Life-Technologies Ampliseq kit,or the Illumina Trueseq kit.). The multiplexed PCR products will now beenriched for mutations in view of the NaME treatment; thus, theresulting library preparation will provide mutation enriched DNA fortargeted re-sequencing.

In some embodiments, prior to implementing the NaME method describedherein, targets of interest are captured from genomic DNA withinmolecular inversion probes (MIPS); or using the ‘bait’ oligonucleotidesapproach. In some embodiments, the MIPS or the bait oligonucleotides arebiotinylated (e.g., similar to those included in the Agilent SureSelect™kit, but re-designed to capture both top and bottom target DNA strandsas per previous sections). In some embodiments, the baitoligonucleotides are attached to beads. The nucleic acid sample iscontacted with the bait oligonucleotides that bind to selected targetsof interest and binding of the bait oligonucleotides to the targets ofinterest is enabled. Next the bait oligonucleotides with the regions ofinterest bound thereto are isolated from the remaining nucleic acids.Finally, the isolated targets are released from beads and multiplexedNaME (using overlapping probes addressing both top and bottom DNAstrands and annealing to numerous targeted gene mutationssimultaneously) is used to cleave the different wild-type nucleic acidssimultaneously. Following this, PCR and library construction using themutation-enriched sample can be used prior to MPS.

In some embodiments, the probe oligonucleotides that can be used in themethods described herein systematically tile a genomic region ofinterest, for example, chromosome Y. In some embodiments, degenerateoligonucleotide probes are synthesized that cover all AT-rich regions,all GC rich regions, gene promoters; or CpG islands. Any genomicfraction of interest can be targeted for selective cleavage ‘at will’using multiple overlapping probes targeting both top and bottom strandsand designed as described herein.

Mutation Enrichment Using NaME in the Absence of SubsequentAmplification.

In all embodiments described thus far, in order to produce a sample withenriched mutated target sequences, an amplification step is conductedfollowing application of DSN digestion of the wild-type DNA alleles.Alternatively, another way to enrich the mutated target sequences is toeliminate the wild-type sequences (without amplification).

Thus, in some embodiments, the reaction mixture with cleavedwild-type-probe duplexes and uncleaved target mutant nucleic acids issubjected to a further DNA degradation condition which hydrolyzesenzymatically the DSN-cleaved wild-type-probe duplexes, with thedegradation initiated at the position of the cleavage. The “DNAdegradation condition” includes contacting the reaction mixture withcleaved wild-type-probe duplexes and uncleaved target mutant nucleicacids to an exonuclease (e.g., using exo I or exo III or Klenow fragmentof E. coli DNA polymerase I that digest DNA from the 3′-end) underconditions of optimal exonuclease activity (that is, the temperature, pHion concentrations etc. are maintained to provide optimal enzymeactivity).

In this approach, genomic DNA can either be not fragmented, or it may berandomly fragmented as described in preceding sections. If the DNA isfragmented, the fragmented genomic DNA is first ligated to adaptors thatare resistant to 3′-exonuclease digestion (e.g., by using adaptors thathave a 3′-terminal phosphorothioate linkage). Next, the DNA sample isdenatured, and NaME is applied as described in previous sections togenerate cleavage of wild-type sequences while leaving intact themutated sequences. Next, an exonuclease digestion is applied (e.g.,using exo I or exo III or Klenow fragment of E. coli DNA polymerase Ithat digest DNA from the 3′-end). Exonuclease will digest all sequencesthat do not have an exonuclease resistant 3′-end, i.e., without3′-terminal phosphorothioate. Since DSN-nicked fragments do not have a3′-terminal phosphorothioate, they will be fully digested by the enzyme,thereby eliminating wild-type DNA strands. Digestion will proceed fromthe 3′-position of the DSN-induced nick all the way to the 5′end, whileleaving the (un-nicked) mutated target sequences intact. Optionally, onemay also digest wild-type sequences from the 5′-end of the nick all theway to the 3′-end by using E. coli DNA polymerase I that has 5′ to 3′exonuclease activity. Following the complete digestion of theDSN-nicked, wild-type sequences, an endpoint detection method that doesnot require amplification, such as a single molecule sequencing approach(Nanopore system; or Pacific-Bio system) can be used to sequence themutation-enriched DNA sample. This embodiment that does not rely on anyform of nucleic acid amplification and can be particularly useful in thesequencing of small genomes (e.g., bacterial or viral genomes) where lowlevel mutations are currently difficult to detect in view of therelatively high error rate of these sequencing systems. The approach canbe combined with selective capture of genomic fragments on beads, toreduce the complexity of a larger genome followed by NaME to eliminatewild-type DNA and enrich mutated target sequences in the absence ofamplification.

Mutation Scanning Using NaME

Often there is a need to scan for mutations in long DNA regions (asopposed to identifying known mutations at a single hotspot position,such as KRAS). Using two longer probes that are complementary toadjacent DNA sequences (one probe on bottom strand and the second,adjacent probe, on the top strand) one can adapt NaME for ‘mutationscanning’. For example, probes of 50-70 bp can be used to cover a regionof 100-140 base pairs. If there is a mutation at any position alongthese 140 base pairs, the corresponding strand will not be substantiallycleaved by DSN. Hence, the mutant strand will survive and will lead to asubsequent PCR product that can be sequenced (FIG. 12A). In this way,longer regions on tumor suppressor genes like p53 can be enriched formutations irrespective of the mutation position (FIGS. 12B and 12C).

NaME Application with RNA or ssDNA

In some embodiments, the methods described herein can be used toselectively cleave wild-type cDNA, or mRNA or DNA in single strandedformat. For this approach (FIG. 13 ), only a single probe per targetedgene would be needed, as opposed to one probe on each opposite strandused in dsDNA.

Methylation-Sensitive NaME (with Probes)

In some embodiments, the methods described herein are used to prepare anunmethylated target nucleic acid of interest for subsequent enrichment.In such embodiments, prior to implementing NaME on the reaction mixture,the nucleic acid sample is treated with sodium bisulfite (bisulfiteconverts all cytosines to uracils, unless the cytosines are methylatedat CpG dinucleotide positions). The pair of oligonucleotide probes usedin these methods are complementary to top and bottom strands of themethylated nucleic acid of interest, that is, one of the oligonucleotideprobes is complementary to top strand of the methylated nucleic acid ofinterest, while the other oligonucleotide probe is complementary to thebottom strand of the methylated nucleic acid of interest followingbisulfite conversion. The probes will, thus, form complementary (i.e.,without any mismatches) duplexes with the top and bottom strands inalleles that contain fully methylated DNA. In contrast, the probes willform partially complementary duplexes with the alleles containingunmethylated cytosines because of mismatches at the positions of uracils(which used to be cytosines before bisulfite conversion). As a result,the methylated duplexes will be cleaved by DSN, while the unmethylatedduplexes will remain substantially intact for subsequent amplification(FIG. 14 ).

Since NaME works well with mismatches caused by single point mutations,it can be expected that presence of several mismatches on a sequence dueto conversion of multiple cytosines makes DSN match/mismatchdiscrimination work even better. Thus, one can optionally also useprobes that are longer, (for example, 50-200 bp or longer) with thisapproach. Furthermore, in contrast to regular double stranded DNA,bisulfite converted DNA remains single stranded after chemicaltreatment, and the two DNA strands are not complementary to each otherany longer. Accordingly, one may optionally use probes matching only thetop DNA strand or matching only the bottom DNA strand followingbisulfate conversion of DNA.

One can use thousands of probes covering all promoters and tiling entiregenomic regions. For example, genomic DNA is digested into smallerfragments, using physical shearing for random fragmentation orrestriction enzyme fragmentation (using enzymes that aremethylation—independent). DNA is randomly fragmented, end repaired, andligated to methylated adaptors that are resistant to bisulfiteconversion. This is a standard first step in whole genome bisulfitesequencing preparations. Next, the sample is treated with sodiumbisulfite, to convert unmethylated C to U (FIG. 14 ). The DNA at thispoint comprises mostly single stranded sequences. Next, the NaMEprocedure is applied, by adding DSN plus a large set of synthesizedoligonucleotide probes designed to match the methylatedbisulfite-converted version of the regions of interest (for example, anentire tumor suppressor gene like TP53 or BRCA1; or a large portion ofchromosome 21 if trisomy 21 is under examination for pre-nataldiagnostics; or all promoters in oncogenes; or regions that aredifferentially methylated among various tissues in order to assistdefinition of the tissue of origin when examining circulating DNA orother liquid biopsies). The probes will form perfectly double strandedDNA (i.e., complementary duplexes) in alleles that contained fullymethylated DNA. Both top and bottom strands of the original DNA need tobe addressed by the oligonucleotides used, as both parent DNA strandsneed to be selectively digested and prevented from subsequentamplification. Alleles with unmethylated DNA will remain undigested,because the probes will contain mismatches at the positions of uracils(bisulfite converted cytosines). As a result, DSN will not cut thesesequences, thereby allowing their subsequent amplification (FIG. 14 ).Following selective cleavage of methylated targets of interest, anamplification condition, such as PCR using the common adaptors, can beapplied followed by sequencing of the sample. Alternatively, one canapply PCR of repeat elements using primers specific forbisulfite-treated ALU, LINE 1 and other repeat elements spread over thegenome; or arbitrarily primed PCR (AP-PCR); or COLD-PCR. Isothermalforms of amplification may also be used in the place of PCR. Thisapproach is of relevance to many applications such as cancerdiagnosis/therapy (for detecting global hypomethylation), to prenataldiagnosis (e.g., for detecting placental DNA which contains fetalsequences that are substantially unmethylated), and to other diseasesknown to result/cause hypomethylation, such as systemic lupuserythymatosis.

In some embodiments, e.g., following sodium bisulfite treatment, themethod is applied in the opposite manner, that is, to prepare amethylated target nucleic acid of interest for subsequent enrichment. Insuch embodiments, the pair of oligonucleotide probes used are fullycomplementary to top and bottom strands of the unmethylated nucleic acidof interest, that is, one of the oligonucleotide probes is complementaryto top strand of the unmethylated nucleic acid of interest, while theother oligonucleotide probe is complementary to the bottom strand of theunmethylated nucleic acid of interest following its bisulfiteconversion. This results in the preferential removal of the unmethylatedregions of the targets of interest.

In some embodiments, e.g., following bisulfite treatment, the method isused to prepare both an unmethylated target nucleic acid of interest anda (different) methylated target nucleic acid of interest for subsequentenrichment. The method comprises: (i) a pair of oligonucleotide probes,one of which is complementary to top strand of the methylated form ofthe unmethylated target nucleic acid of interest, while the other iscomplementary to the bottom strand of the methylated form of theunmethylated target nucleic acid of interest, (ii) a pair ofoligonucleotide probes, one of which is complementary to top strand ofthe unmethylated form of the methylated target nucleic acid of interest,while the other is complementary to the bottom strand of theunmethylated form of the methylated target nucleic acid of interest; andwherein prior to implementing NaME protocol described herein on thereaction mixture, the nucleic acid sample is treated with sodiumbisulfite.

In some embodiments, the method is used to prepare multiple targetnucleic acids of interest, some of which are methylated target nucleicacids of interest, and some of which are unmethylated target nucleicacids of interest. In such embodiments, (i) a pair of oligonucleotideprobes, one of which is complementary to top strand of the methylatedform of each unmethylated target nucleic acid of interest, while theother is complementary to the bottom strand of the methylated form ofeach unmethylated target nucleic acid of interest, and (ii) a pair ofoligonucleotide probes, one of which is complementary to top strand ofthe unmethylated form of each methylated target nucleic acid ofinterest, while the other is complementary to the bottom strand of theunmethylated form of each methylated target nucleic acid of interest areused. Prior to implementing NaME on the reaction mixture, the nucleicacid sample is treated with sodium bisulfite. Thus, the methodsdescribed herein allow for the simultaneously removal of (a)unmethylated promoters in tumor suppressor genes (so that it becomeseasy to reveal the methylated genes of interest), and (b) methylatedpromoters of oncogenes (so that it becomes easy to reveal theunmethylated genes of interest). In this way one can enrich formethylated oncogene promoters, as well as for unmethylated oncogenepromoters, simultaneously.

Finally, similar approaches to those described above using bisulfitetreatment of DNA to selectively enrich 5-methylcytosine (5mC)-baseddifferentially methylated/unmethylated DNA regions may also be appliedto selectively enrich different epigenetic DNA modifications ofinterest, such as 5-hydroxy-methylation (5hmC). 5-hydroxy-methylation isan epigenetic modification functionally and biologically different from5-methylcytosine-modification. Thus, it is important to measureseparately these two modifications. One way to separate DNA containing5hmC from that containing 5mC is TAB-seq (tet-assisted bisulfitesequencing, Ito S et al, Science 2011 333(6047):1300-1303; and Yu M etal, Cell 2012: 149:1368-80). In TAB-seq, genomic 5hmC is first protectedwith protected by glucosylation, prior to performing bisulfiteconversion. The DNA is then treated with a Tet enzyme, converting 5mC to5caC, while leaving the glycosylated 5-hydroxy-methylation untouched.Any C's read in the resulting sequencing are thus interpreted as5-hydroxy-methylated. Accordingly, depending on whether bisulfitetreatment is applied directly, OR following glucosylation, the presentinvention can be directed to enriching either5-methylcytosine-containing DNA or 5-hydroxy-methylcytosine containingDNA.

Nuclease Chain Reaction

Some aspects of the disclosure relate to a method for preparing a targetmutant nucleic acid for subsequent enrichment relative to a wild-typenucleic acid, using a ‘chain reaction approach’. The method comprisesthe steps of: (a) exposing a nucleic acid sample comprising adouble-stranded wild-type nucleic acid and a double-stranded targetnucleic acid suspected of containing a mutation to a thermostable doublestrand-specific nuclease (DSN) and a pair of oligonucleotide probes, oneof which is complementary to the wild-type nucleic acid top strand andthe other is complementary to the wild-type nucleic acid bottom strand,to create a reaction mixture, wherein at least one of the probesoverlaps a sequence on the target nucleic acid containing the suspectedmutation; (b) subjecting the reaction mixture to a denaturingtemperature to permit denaturation of the wild-type nucleic acid and thetarget mutant nucleic acid; and (c) reducing the temperature to permitrapid hybridization of the probes to their corresponding sequences onthe wild-type and target mutant nucleic acids thereby formingcomplementary wild-type-probe duplexes on top and bottom strands, andpartially complementary target mutant-probe duplexes, wherein the DSNcleaves the complementary wild-type-probe duplexes and but not thepartially complementary target mutant-probe duplexes.

More specifically, in some embodiments, NaME can be applied in atemperature cycling fashion, including successive brief denaturationcycles followed by DSN incubation in the presence of probes (FIG. 9 ).The thermostable DSN is included in the reaction mixture from thebeginning despite the use of denaturing temperature. The temperature israised such that it allows denaturation of the wild-type nucleic acidand the target mutant nucleic acid without destroying the DSN enzymewhich is simultaneously present in the reaction mixture. In someembodiments, this denaturing temperature is 65° C., 70° C., 75° C., 80°C., or 85° C.). In some embodiments, an organic solvent that can lowerthe Tm of the nucleic acids is included in the reaction mixture. Thesolvent lowers the Tm of the nucleic acids, without inhibiting theactivity of DSN. Examples of such solvents include, but are not limitedto DMSO, betaine or formamide. In some embodiments, 10-15% of DMSO isincluded in the reaction mixture.

After briefly denaturing the wild-type and target nucleic acids, thetemperature is lowered to permit hybridization of the probes to theircorresponding sequences on the wild-type and mutant nucleic acids. Thishybridization temperature allows for DSN activity. In some embodiments,this hybridization temperature is 50° C., 55° C., 60° C., 65° C., or 70°C.). Since the oligonucleotide probes are in high excess as compared tothe wild-type and target nucleic acids, they bind to their correspondingsequences and form complementary wild-type-probe duplexes on top andbottom strands, and partially complementary mutant-probe duplexes. Thethermostable DSN then digests the wild-type-probe complementaryduplexes, while the partially complementary mutant-probe duplexes remainintact.

In some embodiments, steps (b) (denaturing step) and (c)(hybridization/DSN incubation step) are repeated for one or more cycles.In some embodiments, these steps are repeated for 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, 40, or 50 cycles. The hybridization/DSN incubationstep is only applied intermittently (for example, 2-4 min) and isinterrupted by another denaturation cycle, followed by lowering thetemperature again for DSN digestion, and so on. In this way it ispossible to maximize the difference between wild-type and mutant nucleicacid cleavage, while still preventing substantial re-hybridization ofthe two parent nucleic acid strands (which would lead to indiscriminatedestruction of the nucleic acid, whether mutant or not).

In some embodiments, the method further comprises enriching the targetmutant nucleic acid relative to the wild-type nucleic acid by subjectingthe reaction mixture with cleaved wild-type-probe duplexes and uncleavedtarget mutant nucleic acids to an amplification condition such as butnot limited to PCR, COLD-PCR, ligation mediated PCR or COLD-PCR usingcommon ligated adaptors, multiplex PCR, and isothermal amplification(such as but not limited to displacement amplification based on phi-29based; or Loop Mediated LAMP amplification; or any other isothermal modeof amplification).

In some embodiments, one of the probes overlaps a sequence on the topstrand of the target nucleic acid containing the mutation, while theother probe overlaps a sequence on the bottom strand of the targetnucleic acid containing the mutation and the two probes partiallyoverlap each other.

In some embodiments, the method is used to prepare two or more differenttarget mutant nucleic acids for subsequent enrichment relative tocorresponding wild-type nucleic acids and the method further comprisesone or more additional pairs of probes directed to the differentwild-type nucleic acids, wherein for each pair of probes, one of theprobes is complementary to the wild-type nucleic acid top strand and theother is complementary to the wild-type nucleic acid bottom strand.

NaME-PCR

Some aspects of the present disclosure provide methods that combine theNaME process with PCR amplification in a single step as shown in FIG. 10. Accordingly, aspects of the disclosure provide a method for enrichinga target mutant nucleic acid, the method comprising the steps of: (a)preparing an amplification reaction mixture comprising: adouble-stranded wild-type nucleic acid, a double-stranded target nucleicacid suspected of containing a mutation, a thermostable doublestrand-specific nuclease (DSN), a pair of oligonucleotide probes, one ofwhich is complementary to the wild-type nucleic acid top strand and theother is complementary to the wild-type nucleic acid bottom strand,wherein at least one of the probes overlaps a sequence on the targetnucleic acid containing the suspected mutation and PCR amplificationcomponents; (b) subjecting the reaction mixture to a denaturingtemperature to permit denaturation of the wild-type nucleic acid and thetarget mutant nucleic acid; (c) reducing the temperature to permithybridization of the probes to their corresponding sequences on thewild-type and target mutant nucleic acids thereby forming complementarywild-type-probe duplexes on top and bottom strands, and partiallycomplementary target mutant-probe duplexes, wherein the DSN cleaves thecomplementary wild-type-probe duplexes but not the partiallycomplementary target mutant-probe duplexes; and (d) subjecting thereaction mixture to an amplification condition thereby enriching theuncleaved target mutant nucleic acid relative to the cleaved wild-typenucleic acid.

In this approach, NaME and PCR are performed in a single tube. All PCRcomponents such as but not limited to primers, dNTPs, polymerase andpolymerase buffer are included together with DSN and oligonucleotideprobes in the reaction mixture. In this manner, the wild-type nucleicacid is successively selectively destroyed by DSN while alsore-synthesized by PCR, so that the total target DNA remains the same orincreases, while at the same time continuously enriching the mutated DNAduring the cycling process. In some embodiments, the method is performedin COLD-PCR format, instead of standard PCR.

DSN is compatible with most PCR buffers used commercially; hence DSNcleavage works in a PCR environment. Since both enzymes, DSN andpolymerase, are thermostable it is possible to operate a combinedreaction in a common thermocycler. The amplification condition appliedin step (d) permits annealing of primer pairs to the wild-type andtarget mutant nucleic acids followed by extension thereby enriching thetarget mutant nucleic acid relative to wild-type.

In some embodiments, steps (b) (denaturing step) and (c)(hybridization/DSN incubation step) are repeated for two or more cyclesbefore executing step (d) (PCR amplification). In some embodiments,these steps are repeated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,or 50 cycles. In some embodiments, steps (b) (denaturing step), (c)(hybridization/DSN incubation step) and (d)(amplification step) arerepeated for two or more cycles. In some embodiments, these steps arerepeated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 cycles.

In some embodiments, the reaction mixture further comprises an organicsolvent that can lower the Tm of the nucleic acids is included in thereaction mixture. The solvent lowers the Tm of the nucleic acids,without inhibiting the activity of DSN. Examples of such solventsinclude, but are not limited to DMSO, betaine or formamide.

The denaturing temperature used is such that it allows denaturation ofthe wild-type nucleic acid and the target mutant nucleic acid withoutdestroying the DSN enzyme which is simultaneously present in thereaction mixture. In some embodiments, this denaturing temperature is65° C., 70° C., 75° C., 80° C., or 85° C. applied for time periods of 1sec, 30 sec, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9min, or 10 min.

In some embodiments, one of the probes overlaps a sequence on the topstrand of the target nucleic acid containing the mutation, while theother probe overlaps a sequence on the bottom strand of the targetnucleic acid containing the mutation and the two probes partiallyoverlap each other. In some embodiments, the probes are modified at the3′ end to prevent polymerase extension and to ensure that the probes donot act as primers during NaME-PCR. In some embodiments, the primersused for PCR amplification have a melting temperature that is below thetemperature applied in step (c). This ensures that the primers do notbind to the nucleic acids during the time DSN is used to selectivelycleave wild-type nucleic acid. For example, the Tm of the primers can be55° C. while the hybridization/DSN incubation step is performed above65° C. where the primers do not interfere.

In some embodiments, the method is used to enrich two or more differenttarget mutant nucleic acids relative to wild-type nucleic acids and themethod further comprises one or more additional pairs of probes directedto the different wild-type nucleic acids, wherein for each pair ofprobes, one of the probes is complementary to the wild-type nucleic acidtop strand and the other is complementary to the wild-type nucleic acidbottom strand.

FIG. 10 depicts a process of sequential DNA synthesis via PCR (duringwhich DSN does not have enough time for substantial cleavage of thenucleic acids, while polymerase acts within seconds to synthesizetemplates) followed by arresting amplification for 10 min at 65° C.during which DSN acts selectively on wild-type nucleic acid. At thistemperature, primers do not bind, and hence polymerase synthesis doesnot proceed. This is then followed by more DNA synthesis via PCR andmore DSN digestion and so on in a sequential process. The end product isDNA comprising mainly mutant DNA. The product can be directly sequencedor analyzed for mutations using other known methods.

FIG. 11 depicts combination of NaME with COLD-PCR (COLD-NaME-PCR) foreven greater enrichment of mutation containing nucleic acid. Conditionsfor COLD-PCR are applied that enable selective amplification of mutationcontaining nucleic acid during PCR, followed by selective cleavage ofwild-type nucleic acid using DSN in sequential single tube reactions.All types of COLD-PCR can be used, including but not limited to, fullCOLD, fast COLD, ICE-COLD PCR or Limited Denaturation Time LDT-COLD-PCR.Methods to perform COLD-PCR are highly compatible with NaME-PCR and havebeen described previously (see, for example, Li J, Wang L, Mamon H,Kulke M H, Berbeco R, Makrigiorgos G M. Nat Med 2008; 14:579-84; MilburyC A, Li J, Makrigiorgos G M. Nucleic Acids Res; 39:e2; Murphy D M,Castellanos-Rizaldos E, Makrigiorgos G M. Clin Chem. 2014 60:1014-6; thecontents of which are incorporated by reference herein).

Methylation-Sensitive NaME—No Probes

Some aspects of the disclosure provide methods for preparingunmethylated or methylated nucleic acids of interest usingtemperature-based preferential enrichment of the alleles of interest ona genome wide level using enzymes such as DSN or exonuclease. In thisapproach, no probes need to be used. Genomic DNA is digested intosmaller fragments. Following end repair and bisulfite-resistant adaptorligation, bisulfite treatment is applied. Now the unmethylated sequencesrevert to a lower Tm in view of the C>T conversion, while methylatedsequences remain at high Tm. Next, PCR is applied using the ligatedadaptors, in order to generate amplified double stranded DNA library.(Alternatively, one can apply PCR of repeat elements using primersspecific for bisulfite-treated ALU, LINE 1 and other repeat elementsspread over the genome, in order to apply the method to repeat sequencesonly; or arbitrarily-primed PCR (AP-PCR) to apply the method to large,arbitrary genomic fractions; or one can apply COLD-PCR. Isothermal formsof amplification may also be used in the place of PCR).

Following amplification, one may use either preferential denaturationapproach, or a preferential hybridization approach to enrichunmethylated sequences. The two approaches are described below.

For preferential denaturation approach, the temperature is raised to alevel of choice that generates partial or complete denaturation of lowerTm sequences. These include the originally unmethylated sequences whichdue to the C>U conversion resulted to a C>T conversion in the final PCRproduct, reverting to a lower Tm than the methylated sequences. High Tmsequences remain double stranded. These include the highly methylatedGC-rich sequences. One of ordinary skill can determine the Tm of thesequences using methods known in the art and as described herein. Thetemperature used for this preferential denaturation of the low Tmsequences includes, but is not limited to 50° C., 55° C. 60° C. 66° C.,70° C., 75° C., 78° C., 80° C., or 85° C.). Next, DSN is added, and thenucleic acids are exposed to conditions optimal for DSN activity.Conditions optimal for DSN activity include the most favorableconditions that allow the enzyme to work most efficiently for cleavingcomplementary duplexes. The optimum DSN activity may be affected byconditions which include temperature; pH; and salt concentrations. Insome embodiments, the temperature used for optimal for DSN activityincludes, but not limited to 50° C., 55° C., 60° C., 65° C., or 70° C.The originally unmethylated sequences with lower Tm, (as well asnaturally AT-rich sequences with lower Tm) will be denatured (i.e., willbecome single stranded) and therefore will escape DSN digestion, whilethe originally methylated sequences will be cleaved since they willremain in double stranded formation at the denaturation temperaturechosen. A subsequent amplification of the remaining sequences using theligated common adaptors will amplify preferentially the intactunmethylated sequences and will enable downstream massively parallelsequencing of unmethylated alleles. In some embodiments, thepreferential denaturation of genomic DNA is performed in the presence oforganic solvents that can lower the Tm of the nucleic acids. Examplesinclude but are not limited to betaine, DMSO, or formamide. It is knownthat betaine generates a narrower melting peak for DNA duplexes, henceby adding betaine the discrimination between high and low Tm sequencesat a given denaturation temperature will be ‘sharper’.

Accordingly, in some embodiments, the method comprises the steps of: (a)ligating bisulfite-resistant adaptors to double stranded nucleic acidsof interest; (b) subjecting the adaptor-linked nucleic acids to sodiumbisulfite treatment and a nucleic acid amplification reaction to formdouble-stranded bisulfite-treated nucleic acids; (c) subjecting thebisulfite-treated nucleic acids to a temperature that permitspreferential denaturation of unmethylated nucleic acids while methylatednucleic acids remain double-stranded; and (d) exposing the unmethylatedand methylated nucleic acids to double strand-specific nuclease (DSN)and conditions for optimal DSN activity, wherein the DSN cleaves themethylated double-stranded nucleic acids but not the unmethylatedsingle-stranded nucleic acids.

Alternatively, for preferential re-hybridization approach, a completedenaturation step is applied. The denaturing temperature should besufficiently high so as to allow the full denaturation of the nucleicacids (e.g., 75° C., 80° C., 85° C., 90° C., or 95° C.). In someembodiments, the denaturing temperature is applied for 30 seconds, 1min, 2 min, or 3 min. In some embodiments, the denaturing temperature of95° C. is applied for 2 min. Following this, the temperature is loweredto a level that allows methylated (or other high Tm) sequences tore-hybridize rapidly (due to their higher Tm), while unmethylatedsequences stay substantially single stranded. Next, application of DSNenzyme at conditions for optimal DSN activity digests preferentially themethylated duplexes. In some embodiments, the re-hybridization takesplace in the presence of an organic solvent such as DMSO which lowersthe Tm of the nucleic acid in combination with concurrent digestionusing DSN (that is, instead of adding DSN in a later step). Use oforganic solvents such as DMSO allows temperatures compatible with DSNaction (e.g., 60-75° C.) to be applied during re-hybridization. Finally,a PCR amplification step can be applied to amplify preferentially thenon-digested, unmethylated alleles followed by sequencing.

Accordingly, in some embodiments, the method comprises the steps of: (a)ligating bisulfite-resistant adaptors to double stranded nucleic acidsof interest; (b) subjecting the adaptor-linked nucleic acids to sodiumbisulfite treatment and a nucleic acid amplification reaction to formdouble-stranded bisulfite-treated nucleic acids; (c) subjecting thebisulfite-treated nucleic acids to a denaturing temperature that permitsdenaturation of both unmethylated and methylated nucleic acids to formunmethylated and methylated single stranded nucleic acids; (d) reducingthe temperature to permit preferential formation of methylated duplexes,but not unmethylated duplexes; and (d) exposing the unmethylated andmethylated nucleic acids to double strand-specific nuclease (DSN) andconditions for optimal DSN activity, wherein the DSN preferentiallycleaves the methylated duplexes but not the unmethylated single-strandednucleic acids.

Bisulfite conversion of DNA has formed the basis of identifying themethylation state of individual genes. With the advent of highthroughput parallel sequencing methods, this technology has extended tothe sequencing of libraries of bisulfite-treated DNA. The approachinvolves fragmenting DNA, ligating adaptors, bisulfite treatment andthen amplifying the libraries for high throughput sequencing (see, forexample, US 2013/0059734, US 2008/0254453, US 2009/0148842 and U.S. Pat.No. 8,440,404).

In a reverse approach that aims to enrich the methylated alleles,following preferential denaturation of the lower Tm sequences (whichincludes unmethylated alleles), treatment with any enzyme with selectiveaction against single stranded (as opposed to double stranded) DNA suchas but not limited to exonuclease I, or III is applied to remove thesingle stranded sequences. Subsequently, the remaining sequences(including the substantially methylated alleles) can be amplified andsequenced. Accordingly, in some embodiments, the method comprises thesteps of: (a) ligating bisulfite-resistant adaptors to double strandednucleic acids of interest; (b) subjecting the adaptor-linked nucleicacids to sodium bisulfite treatment and a nucleic acid amplificationreaction to form double-stranded bisulfite-treated nucleic acids; (c)subjecting the bisulfite-treated nucleic acids to a temperature thatpermits preferential denaturation of unmethylated nucleic acids whilemethylated nucleic acids remain double-stranded; and (d) exposing theunmethylated and methylated nucleic acids to an exonuclease andconditions for optimal exonuclease activity, wherein the exonucleasecleaves the unmethylated single-stranded nucleic acids but not themethylated double-stranded nucleic acids.

In some embodiments, the method comprises the steps of: (a) ligatingbisulfite-resistant adaptors to double stranded nucleic acids ofinterest; (b) subjecting the adaptor-linked nucleic acids to sodiumbisulfite treatment and a nucleic acid amplification reaction to formdouble-stranded bisulfate-treated nucleic acids; (c) subjecting thebisulfate-treated nucleic acids to a denaturing temperature that permitsdenaturation of both unmethylated and methylated nucleic acids to formunmethylated and methylated single stranded nucleic acids; (d) reducingthe temperature to permit preferential formation of methylated duplexes,but not unmethylated duplexes; and (d) exposing the unmethylated andmethylated nucleic acids to an exonuclease and conditions for optimalexonuclease activity, wherein the exonuclease preferentially cleaves theunmethylated single-stranded nucleic acids, but not the methylatedduplexes.

In some embodiments, the nucleic acid amplification reaction used toform double-stranded bisulfite-treated nucleic acids is selected fromthe group consisting of: PCR; full COLD-PCR, fast COLD-PCR;ice-COLD-PCR; temperature-tolerant COLD-PCR; LDT-COLD-PCR; AP-PCR; andrepeat element PCR (ALU, LINE1, and other repeat sequences). In someembodiments, the resultant cleaved unmethylated single stranded nucleicacids and the uncleaved methylated duplexes are subjected to anamplification condition using the bisulfite resistant ligated adaptors.In some embodiments, the amplification condition is selected from thegroup consisting of: PCR; LDT-COLD-PCR; AP-PCR; and repeat element PCR(ALU, LINE1, and other repeat sequences).

The specific advantage of performing the genome-wide amplification inCOLD-PCR format is that, by employing a desired denaturation temperatureduring amplification, COLD-PCR provides an additional enrichment oflower-Tm sequences, as has been demonstrated previously by usingCOLD-PCR on unmethylated single gene sequences (Castellanos-Rizaldos,E., Milbury, C. A., Karatza, E., Chen, C. C., Makrigiorgos, G. M. andMerewood, A. (2014) COLD-PCR amplification of bisulfite-converted DNAallows the enrichment and sequencing of rare un-methylated genomicregions. PLoS One, 9, e94103.). Sequences with Tm above the selecteddenaturation temperature do not amplify during COLD-PCR. Any of thedescribed COLD-PCR formats can be used to amplify selected fractions ofun-methylated sequences from the genome (full COLD-PCR (11); fastCOLD-PCR (11); ice-COLD-PCR (12); temperature-tolerant COLD-PCR (13);and limited denaturation time LDT-COLD-PCR, Murphy D M,Castellanos-Rizaldos E, Makrigiorgos G M. Clin Chem. 2014 60:1014-6).Each COLD-PCR format will achieve amplification of different genomicfractions. For example, fast COLD-PCR utilizes a single denaturationtemperature, hence any sequence with Tm above this temperature will notbe amplified at all. Alternatively, temperature tolerant COLD-PCRutilizes successive steps of increasing denaturation temperaturescovering a broad range of temperatures (e.g., a range of 10° C. in 10steps of 1° C. each), hence amplifying a broader range of unmethylatedsequences from the genome. Depending on the application, differentCOLD-PCR programs can be employed. FIG. 15 describes the combination ofDSN digestion of methylated sequences with tandem temperature tolerantCOLD-PCR amplification of unmethylated sequences for further enrichmentof globally unmethylated alleles.

In some embodiments of the methods used for preparing unmethylated ormethylated nucleic acids of interest without using oligonucleotideprobes, the naturally AT-rich sequences are removed prior to the sodiumbisulfite treatment. Specifically, in the approaches described hereinfor preferential amplification of un-methylated DNA (or methylated DNA),a bisulfite step is applied that converts sequences that were originallyat higher Tm (GC-rich sequences that are un-methylated) to lower Tmsequences (due to the C>T conversion). In contrast, methylated allelesretain their high Tm. In this way, a preferential denaturation stepfollowed by DSN digestion and by subsequent amplification preferentiallyenriches the unmethylated alleles. However, during this amplificationstep, sequences with naturally low Tm will also be amplifiedirrespective of their methylation status (for example, AT-richsequences). In some embodiments, such sequences with melting temperature(Tm) below a temperature of choice are removed to avoid these multiple‘normal, non-informative’ sequences with low Tm to amplify incompetition to the target sequences, thereby overwhelming theamplification of the sequences of interest.

Such ‘non-informative’ sequences can be substantially depleted from thesample before performing the bisulfite conversion step as follows:

-   -   (a) Removing lower—Tm sequences. A temperature-based        fractionation of the starting material is performed just prior        to sodium bisulfite treatment (FIG. 16 ). Following shearing of        genomic DNA, the DNA is treated with an enzyme that creates        blunt ends (e.g., ‘end repair’) such as but not limited to T7        DNA polymerase. Next, the temperature is raised to a desired        cutoff temperature. This desired cut off temperature is the        temperature at which sequences having a lower Tm need to be        selectively removed. As an example, let it be assumed that the        desired cutoff temperature is 80° C. By raising the temperature        to 80° C. for time periods of 1 sec-5 min (e.g., 1 sec, 5 secs,        15 secs, 30 secs, 45 secs, 1 min, 2 mins, 3 mins, 4, mins, or 5        mins), DNA fragments with Tm below 80° C. will be substantially        denatured, while other fragments with Tm above 80° C. will        remain substantially double stranded. Next, the temperature is        quickly lowered, for example, by placing the sample on ice.        Next, an enzyme that degrades preferentially single stranded DNA        is added (for example, exonucleases I or III) and the        temperature is raised to the optimal temperature for this enzyme        activity (for example, 37° C.) for 1 min-60 min (e.g., 1 min, 5        mins, 10 mins, 20 mins, 30 mins, 40 mins, 45 mins, 50 mins, 55        mins, or 60 mins). Due to the complexity of genomic DNA, during        this time period there is no substantial re-naturation of the        single stranded fragments that undergo denaturation, and these        become degraded by the enzyme. Finally, the exonuclease is heat        inactivated. This process yields double stranded DNA fragments        with Tm higher than about 80° C. In this way, fragments with Tm        substantially lower than the chosen cutoff temperature are        substantially depleted. In some embodiments, the process is        repeated for additional rounds if more strict temperature        fractionation is needed. The additional rounds can be at the        same temperature (for example, 80° C.) or different temperatures        (for example, 82-85° C.). Following removal of sequences with Tm        below the desired cut off temperature, a sodium bisulfite step        is applied to selectively convert C to U in unmethylated CpG        positions in nucleic acids. Accordingly, these unmethylated DNA        fragments will now revert to sequences with lower Tm. Because        the majority of sequences with naturally lower Tm (for example,        AT-rich sequences) has been removed during exonuclease-based        temperature fractionation, it is now possible to remove higher        Tm methylated sequences by preferential DSN digestion of        double-stranded sequences, as well to amplify preferentially the        unmethylated sequences via an amplification step such as PCR (or        COLD-PCR) without interference of the lower Tm AT-rich        sequences.    -   (b) Solid support-based temperature fractionation. Another        approach for removing sequences with selected Tm from a genomic        DNA sample includes solid support-based temperature        fractionation. In some embodiments, the solid support comprises        magnetic beads. Magnetic beads may be used for immobilization of        the genomic DNA sample in order to enable separation of genomic        DNA fragments into discrete temperature domains prior to further        treatment (FIG. 17 ). After separating the genomic DNA fragments        into discrete temperature domains, preferential amplification of        unmethylated alleles can be applied separately within each        domain, with minimal interference from amplification of        non-desired lower Tm DNA fragments that do not provide any        enrichment advantage. To apply this protocol, the ligation step        depicted in FIG. 15 is performed with a mix of biotinylated and        non-biotinylated bisulfite-resistant adaptors following which        the majority of genomic DNA fragments are captured on        streptavidin-coated magnetic beads from one strand only (the        opposite strand remaining non-biotinylated). The conditions        applied (total beads relative to total DNA) enable        immobilization of several hundred nanograms of biotinylated DNA        so that enough sequence copies are immobilized to retain        low-level events in the fragment population. Following washing        of unbound DNA, the temperature is ramped-up in 5 different        consecutive steps differing by, for example, 3° C. During each        step, the non-biotinylated DNA strands from lower-Tm fragments        are gradually denaturing and are eluted in the supernatant which        are then collected following bead magnetization (FIG. 17 ). DNA        transitions from double stranded to mostly single stranded        within an interval of ˜4-5° C. By collecting the supernatant in        temperature intervals of 3° C., the genomic DNA fragments are        highly enriched (for example, 10-100-fold) in sequences within        pre-defined temperature domains.

No DSN

Some aspects of the disclosure provide a method for preparing a targetmutant nucleic acid for subsequent enrichment relative to a wild-typenucleic acid comprising subjecting a nucleic acid sample comprising adouble-stranded wild-type nucleic acid and a double-stranded targetnucleic acid suspected of containing a mutation to a condition thatdestabilizes the double stranded wild-type and target mutant nucleicacids; contacting the destabilized double stranded wild-type and targetmutant nucleic acids with a pair of oligonucleotide probes, one of whichis complementary to the wild-type nucleic acid top strand and the otheris complementary to the wild-type nucleic acid bottom strand, to permithybridization of the probes to their corresponding sequences on thewild-type and target mutant nucleic acids thereby forming complementarywild-type-probe duplexes on top and bottom strands, and partiallycomplementary target mutant-probe duplexes, wherein at least one of theprobes overlaps a sequence on the target nucleic acid containing thesuspected mutation, and wherein one or both probes comprise a lockednucleic acid (LNA), peptide nucleic acid (PNA), xeno nucleic acid (XNA),or a nucleic acid with any known modified base or RNA which is capableof blocking PCR amplification; and subjecting the complementarywild-type-probe duplexes on top and bottom strands, and partiallycomplementary target mutant-probe duplexes to an amplificationcondition. The probes that overlap the mutation position act to blockPCR amplification, e.g., acting as a clamp, for the wild-type top andbottom DNA strands, thereby inhibiting amplification of the wild-typenucleic acid. When the probe duplexes with a partially complementarytarget mutant sequence, it is less able to inhibit PCR amplification,thereby permitting selective amplification of the mutant nucleic acid ascompared to the wild-type, without a need for a cleaving enzyme (e.g.,DSN).

The present invention is further illustrated by the following Example,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

Example

NaME on Double-Stranded DNA

NaME utilizes nucleases (DNases) that have a substantially higheractivity on double-stranded DNA (ds DNA) versus single-stranded DNA (ssDNA). Many DNases display such activity, including native shrimpdsDNase, recombinant shrimp dsDNase, King crab nuclease (DSN) and bovineDNase I. In the following sections, NaME embodiments for DSNthermostable nuclease are provided, but the same approaches can be usedfor all other nucleases that display substantially higher activity fords DNA versus ss DNA. Thermostable nuclease (DSN) selectively degradesdouble stranded DNA (or DNA/RNA hybrids), while it has minimal or noaction on single stranded DNA or RNA.

For the purposes of the present disclosure, the term “double-strandspecific nuclease” or “DSN” includes DNA/RNA guided enzymes which havepreferential activity on double-stranded DNA, as opposed to singlestranded DNA. Examples of such enzymes that can be employed inconjunction with NaME include the RNA-guided Cas9 enzymes (Gu et al,Depletion of Abundant Sequences by Hybridization (DASH): Using Cas9 toremove unwanted high-abundance species in sequencing libraries andmolecular counting applications Genome Biology 2016; 17, 41), or theArgonaute DNA-guided enzymes (Gao et al, DNA-guided genome editing usingthe Natronobacterium gregoryi Argonaute, Nature Biotechnology May 2016advanced online publication). These DNA/RNA guided enzymes digest DNAwith high preference when the probe (‘guide oligonucleotide’) is fullymatched to the target DNA, and less so when there is a mismatch. Byemploying probes targeting both top and bottom DNA strands in anoverlapping fashion as described in the present invention, NAME can beapplied with DNA/RNA guided enzymes, in the same manner as when usingother DSN nucleases described herein.

NaME takes advantage of the DSN properties to degrade specific sequencesfrom both the top and bottom DNA strands of wild-type (WT) DNA (FIG.1A). In contrast, mutation-containing DNA is not degraded or degradedmuch less than the WT DNA. Hence, a subsequent PCR reaction after DSNdigestion specifically amplifies the mutant alleles that remainsubstantially intact.

An example of the application of this approach is demonstrated in FIG.1B: a 114 bp ds KRAS PCR amplicon with a 5% mutation was subjected tothe process of FIG. 1A. The DNA template used consisted of the KRAS PCRamplicon with a 5% mutation (1:1000, 1:10,000 (approximately 0.001 nM),and 1:100,000) and wtDAN-Taqman-probe (500 nM) and with or withoutKRAS-cutter (500 nM). The samples were then either incubated with DSN (1U) or without DSN. For the 1:1000 and 1:100,000 samples, the mixture washeated to 67° C. for 10 minutes and then 94° for 2 minutes. For the1:10,000 samples, the mixture was heated to a selected temperature (63°C., 67° C., 70° C., or 73° C.) for 10 minutes, and then heated to 94° C.for two minutes. By comparing the mutation abundance in parallelreactions without DSN versus reactions with DSN, the data in FIG. 1B andTable 1 demonstrate mutation enrichments of about 10-fold fortemperatures 63-67° C.

TABLE 1 KRAS Mutation Abundance at Different Temperatures Delta Finalmutation Enrichment- Sample ID Ct abundance fold 1-10k-NO-DSN 0 4.14 1.0(untreated) 1-10k-1U-63C 9.07 38.6 9.3 1-10k-1U-67C 8.58 37.4 9.01-10k-1U-70C 6.54 14 3.4 1-10k-1U-73C 7.11 19.5 4.7 1-10k-1U-67C-no-5.75 6.07 1.5 cutter

NaME Applied Directly from Genomic DNA

FIGS. 5-7 demonstrate NaME applied directly to genomic DNA for a singleKRAS target sequence, or single p53 sequence (FIG. 5 ), two targetssimultaneously, duplex KRAS and p53 (FIG. 6 ) and three different KRASmutations in a single-target reaction (FIG. 7A). In FIG. 5 , genomic DNAwith approximately a 0.5% KRAS mutation, or a p53 mutation in a separatereaction was denatured at 95° C. and incubated at 65° C. for 20 minutesin the presence of overlapping blocked probes and DSN. The enrichment ofthe KRAS or p53 mutations were detected via a subsequent digital PCRthat quantifies the mutation abundance. In FIG. 6 , the genomic DNAunderwent the same protocol as in FIG. 5 , except that the KRAS and p53mutated genomic DNA were mixed in a single tube. Genomic DNA from theSW480 cell line containing both KRAS and p53 mutations was mixed withwild-type DNA to create low-abundance mutations on both genes. Twomutation percentages were tested: approximately 5% and approximately0.3%. In FIG. 7A, genomic DNA with 1-5% KRAS mutations from threedifferent cell lines, in separate reactions, underwent the same protocolas described above. In FIG. 7B, the DNA was denatured, the temperaturewas reduced to 67° C., DSN and overlapping probes were added for a20-minute incubation. The DSN was inactivated, and PCR and digital PCRwere performed on each target amplicon to derive their respectivemutational abundances. All mutations are enriched simultaneously viaNaME. FIG. 7B depicts mutation enrichment when NaME is applied on 11targets simultaneously, directly from genomic DNA obtained from HorizonDx, containing known low-level mutations on the respective targets. Itis demonstrated that all 11 targets are enriched simultaneouslyfollowing application of NaME directly to genomic DNA.

Mutation Scanning Using NaME

FIGS. 12B and 12C depict the results when using NaME for mutationenrichment of a 40-80 bp region in TP53 exon 8. All of the 4 mutationstested are enriched via NaME. In FIG. 12B, mutation-containing DNA fromthe PFSK or HCC cell lines was serially diluted into WT DNA, and then afirst PCR was applied to amplify the target of interest (tp53). Theamplicon was denatured and then incubated at 65° C. in the presence ofprobes and DSN. The two probes correspond to the WT top and bottomstrands, respectively. The presence of a mutation inhibits DSNdigestion, hence the mutated DNA is amplified during rounds of PCR. Theeffects of probe length and concentration on mutation enrichment arealso depicted in FIG. 12B. In FIG. 12C, mutation-containing DNA fromPFSK, HCC, SW480, and MDAMB cell lines, all of which have differentmutations on p53 exon 8, were serially diluted into WT DNA, and then thesame protocol as described in FIG. 12B was performed. The mutationenrichment-fold was calculated by performing digital PCR both before andafter NaME application.

REFERENCES

-   1. Thomas, R. K., Baker, A. C., Debiasi, R. M., Winckler, W.,    Laframboise, T., Lin, W. M., Wang, M., Feng, W., Zander, T.,    Macconnaill, L. E. et al. (2007) High-throughput oncogene mutation    profiling in human cancer. Nat Genet, 39, 347-351.-   2. Chou, L. S., Lyon, E. and Wittwer, C. T. (2005) A comparison of    high-resolution melting analysis with denaturing high-performance    liquid chromatography for mutation scanning: cystic fibrosis    transmembrane conductance regulator gene as a model. Am J Clin    Pathol, 124, 330-338.-   3. Thomas, R. K., Nickerson, E., Simons, J. F., Janne, P. A., Tengs,    T., Yuza, Y., Garraway, L. A., Laframboise, T., Lee, J. C., Shah, K.    et al. (2006) Sensitive mutation detection in heterogeneous cancer    specimens by massively parallel picoliter reactor sequencing. Nat    Med, 12, 852-855.-   4. Paez, J. G., Janne, P. A., Lee, J. C., Tracy, S., Greulich, H.,    Gabriel, S., Herman, P., Kaye, F. J., Lindeman, N., Boggon, T. J. et    al. (2004) EGFR Mutations in Lung Cancer: Correlation with Clinical    Response to Gefitinib Therapy. Science, 304, 1497-1500.-   5. Janne, P. A., Borras, A. M., Kuang, Y., Rogers, A. M., Joshi, V.    A., Liyanage, H., Lindeman, N., Lee, J. C., Halmos, B., Maher, E. A.    et al. (2006) A rapid and sensitive enzymatic method for epidermal    growth factor receptor mutation screening. Clin Cancer Res, 12,    751-758.-   6. Engelman, J. A., Mukohara, T., Zejnullahu, K., Lifshits, E.,    Borras, A. M., Gale, C. M., Naumov, G. N., Yeap, B. Y., Jarrell, E.,    Sun, J. et al. (2006) Allelic dilution obscures detection of a    biologically significant resistance mutation in EGFR-amplified lung    cancer. The Journal of clinical investigation.-   7. Diehl, F., Li, M., Dressman, D., He, Y., Shen, D., Szabo, S.,    Diaz, L. A., Jr., Goodman, S. N., David, K. A., Juhl, H. et    al. (2005) Detection and quantification of mutations in the plasma    of patients with colorectal tumors. Proc Natl Acad Sci USA, 102,    16368-16373.-   8. Kimura, T., Holland, W. S., Kawaguchi, T., Williamson, S. K.,    Chansky, K., Crowley, J. J., Doroshow, J. H., Lenz, H. J.,    Gandara, D. R. and Gumerlock, P. H. (2004) Mutant DNA in plasma of    lung cancer patients: potential for monitoring response to therapy.    Annals of the New York Academy of Sciences, 1022, 55-60.-   9. Nilsen, I. W., Overbo, K., Jensen Havdalen, L., Elde, M.,    Gjellesvik, D. R. and Lanes, O. (2010) The enzyme and the cDNA    sequence of a thermolabile and double-strand specific DNase from    Northern shrimps (Pandalus borealis). PLoS One, 5, e10295.-   10. Castellanos-Rizaldos, E., Milbury, C. A., Karatza, E., Chen, C.    C., Makrigiorgos, G. M. and Merewood, A. (2014) COLD-PCR    amplification of bisulfite-converted DNA allows the enrichment and    sequencing of rare un-methylated genomic regions. PLoS One, 9,    e94103.-   11. Li, J., Wang, L., Mamon, H., Kulke, M. H., Berbeco, R. and    Makrigiorgos, G. M. (2008) Replacing PCR with COLD-PCR enriches    variant DNA sequences and redefines the sensitivity of genetic    testing. Nat Med, 14, 579-584.-   12. Milbury, C. A., Li, J. and Makrigiorgos, G. M. (2011)    Ice-COLD-PCR enables rapid amplification and robust enrichment for    low-abundance unknown DNA mutations. Nucleic Acids Res, 39, e2.-   13. Castellanos-Rizaldos, E., Liu, P., Milbury, C. A., Guha, M.,    Brisci, A., Cremonesi, L., Ferrari, M., Mamon, H. and    Makrigiorgos, G. M. (2012) Temperature-Tolerant COLD-PCR Reduces    Temperature Stringency and Enables Robust Mutation Enrichment. Clin    Chem, 58, 1130-1138.-   14. Shagin, D. A., Rebrikov, D. V., Kozhemyako, V. B., Altshuler, I.    M., Shcheglov, A. S., Zhulidov, P. A., Bogdanova, E. A.,    Staroverov, D. B., Rasskazov, V. A. and Lukyanov, S. (2002) A novel    method for SNP detection using a new duplex-specific nuclease from    crab hepatopancreas. Genome Res, 12, 1935-1942.-   15. Qiu, X., Zhang, H., Yu, H., Jiang, T. and Luo, Y. (2015)    Duplex-specific nuclease-mediated bioanalysis. Trends in    biotechnology, 33, 180-188.

What is claimed is:
 1. A method for enriching a target mutant nucleicacid, comprising: (a) preparing an amplification reaction mixturecomprising: a double-stranded wild-type nucleic acid, a double-strandedtarget nucleic acid suspected of containing a mutation, a thermostabledouble strand-specific nuclease (DSN), a pair of oligonucleotide probes,one of which is complementary to the wild-type nucleic acid top strandand the other is complementary to the wild-type nucleic acid bottomstrand, wherein at least one of the probes overlaps a sequence on thetarget nucleic acid containing the suspected mutation and PCRamplification components and the two probes only partially overlap eachother; (b) subjecting the reaction mixture to a denaturing temperatureto permit denaturation of the wild-type nucleic acid and the targetmutant nucleic acid; (c) reducing the temperature to permithybridization of the probes to their corresponding sequences on thewild-type and target mutant nucleic acids thereby forming complementarywild-type-probe duplexes on top and bottom strands, and partiallycomplementary target mutant-probe duplexes, wherein the DSN cleaves thecomplementary wild-type-probe duplexes but not the partiallycomplementary target mutant-probe duplexes; and (d) subjecting thereaction mixture to an amplification condition thereby enriching theuncleaved target mutant nucleic acid relative to the cleaved wild-typenucleic acid.
 2. The method of claim 1, further comprising repeatingsteps (b) and (c) for two or more cycles before executing step (d). 3.The method of claim 2, further comprising repeating steps (b), (c) and(d) for two or more cycles.
 4. The method of claim 1, wherein thereaction mixture further comprises an organic solvent.
 5. The method ofclaim 1, wherein the denaturing temperature is between 65-85° C.
 6. Themethod of claim 1, wherein both probes overlap the suspected mutation.7. The method of claim 1, wherein each probe is modified at the 3′ endto prevent polymerase extension.
 8. The method of claim 1, whereinprimers used for PCR amplification have a melting temperature that isbelow the temperature applied in step (c).
 9. The method of claim 1,wherein the method is used to enrich two or more different target mutantnucleic acids relative to wild-type nucleic acids and the method furthercomprises one or more additional pairs of probes directed to thedifferent wild-type nucleic acids, wherein for each pair of probes, oneof the probes is complementary to the wild-type nucleic acid top strandand the other is complementary to the wild-type nucleic acid bottomstrand.
 10. The method of claim 1, wherein the amplification conditionis PCR; full COLD-PCR, fast COLD-PCR; ice-COLD-PCR, temperature-tolerantCOLD-PCR and limited denaturation time COLD-PCR.
 11. A method forpreparing unmethylated nucleic acids of interest for subsequentenrichment relative to corresponding methylated nucleic acidscomprising: (a) ligating bisulfite-resistant adaptors to double strandednucleic acids of interest; (b) subjecting the adaptor-linked nucleicacids to sodium bisulfite treatment and a nucleic acid amplificationreaction to form double-stranded bisulfite-treated nucleic acids; (c)subjecting the bisulfite-treated nucleic acids to a temperature thatpermits preferential denaturation of unmethylated nucleic acids whilemethylated nucleic acids remain double-stranded or denaturation of bothunmethylated and methylated nucleic acids to form unmethylated andmethylated single stranded nucleic acids; (d) exposing the unmethylatedand methylated nucleic acids to double strand-specific nuclease (DSN) oran exonuclease.
 12. The method of claim 11, wherein the temperaturepermits preferential denaturation of unmethylated nucleic acids whilemethylated nucleic acids remain double-stranded.
 13. The method of claim12, wherein the unmethylated and methylated nucleic acids are exposed todouble strand-specific nuclease (DSN) and conditions for optimal DSNactivity, wherein the DSN cleaves the methylated double-stranded nucleicacids but not the unmethylated single-stranded nucleic acids.
 14. Themethod of claim 12, wherein the unmethylated and methylated nucleicacids are exposed to an exonuclease and conditions for optimalexonuclease activity, wherein the exonuclease cleaves the unmethylatedsingle-stranded nucleic acids but not the methylated double-strandednucleic acids.
 15. The method of claim 11, wherein the temperaturepermits denaturation of both unmethylated and methylated nucleic acidsto form unmethylated and methylated single stranded nucleic acids. 16.The method of claim 15, further comprising reducing the temperature topermit preferential formation of methylated duplexes, but notunmethylated duplexes.
 17. The method of claim 16, wherein theunmethylated and methylated nucleic acids are exposed to doublestrand-specific nuclease (DSN) and conditions for optimal DSN activity,wherein the DSN preferentially cleaves the methylated duplexes but notthe unmethylated single-stranded nucleic acids.
 18. The method of claim16, wherein the unmethylated and methylated nucleic acids are exposed toan exonuclease and conditions for optimal exonuclease activity, whereinthe exonuclease cleaves the unmethylated single-stranded nucleic acidsbut not the methylated double-stranded nucleic acids.
 19. The method ofclaim 11, wherein the nucleic acid amplification reaction of step (b) isselected from the group consisting of: PCR; full COLD-PCR, fastCOLD-PCR; ice-COLD-PCR, temperature-tolerant COLD-PCR and limiteddenaturation time COLD-PCR.
 20. The method of claim 14, wherein thecleaved unmethylated single stranded nucleic acids and the uncleavedmethylated duplexes are subjected to an amplification condition usingthe bisulfite resistant adaptors ligated in step (a).
 21. The method ofclaim 18, wherein the cleaved unmethylated single stranded nucleic acidsand the uncleaved methylated duplexes are subjected to an amplificationcondition using the bisulfite resistant adaptors ligated in step (a).22. The method of claim 11, wherein naturally AT-rich sequences areremoved prior to the sodium bisulfite treatment.